Regulation of human transmembrane serine protease

ABSTRACT

Reagents that regulate human transmembrane serine protease activity and reagents that bind to human transmembrane serine protease gene products can be used to regulate extracellular matrix degradation. Such regulation is particularly useful for treating COPD, metastasis of malignant cells, tumor angiogenesis, inflammation, atherosclerosis, neurodegenerative diseases, and pathogenic infections.

[0001] This application claims the benefit of and incorporates byreference co-pending provisional applications Ser. No. 60/211,224 filedJun. 13, 2000, Ser. No. 60/283,353 filed Apr. 13, 2001, and Ser. No.60/283,648 filed Apr. 16, 2001, and PCT application ______ filed Jun.12, 2001 under Attorney Docket No. LIO-81-WO.

TECHNICAL FIELD OF THE INVENTION

[0002] The invention relates to the area of regulation humantransmembrane serine protease activity to provide therapeutic effects.

BACKGROUND OF THE INVENTION

[0003] Metastasizing cancer cells invade the extracellular matrix usingplasma membrane protrusions that contact and dissolve the matrix withproteases. Agents that inhibit such protease activity can be used tosuppress metastases. Proteases also are expressed during development,when degradation of the extracellular matrix is desired. In cases whereappropriate extracellular matrix degradation does not occur, supplying amolecule with a protease activity can provide the necessary enzymaticactivity. Thus, there is a need in the art for identifying new proteasesand methods of regulating extracellular matrix degradation.

SUMMARY OF THE INVENTION

[0004] It is an object of the invention to provide reagents and methodsof regulating human transmembrane serine protease. These and otherobjects of the invention are provided by one or more of the embodimentsdescribed below.

[0005] One embodiment of the invention is a cDNA encoding a polypeptidecomprising an amino acid sequence selected from the group consisting of(a) the amino acid sequence shown in SEQ ID NO:12, (b) the amino acidsequence encoded by a cDNA insert contained within plasmid pCRII-TMSP3(ATCC Accession No. ______), and (c) biologically active variantsthereof.

[0006] Yet another embodiment of the invention is an expression vectorcomprising a polynucleotide which encodes a polypeptide comprising anamino acid sequence selected from the group consisting of (a) the aminoacid sequence shown in SEQ ID NO:12, (b) the amino acid sequence encodedby a cDNA insert contained within plasmid pCRII-TMSP3 (ATCC AccessionNo. ______), and (c) biologically active variants thereof.

[0007] Another embodiment of the invention is a host cell comprising anexpression vector which encodes a polypeptide comprising an amino acidsequence selected from the group consisting of (a) the amino acidsequence shown in SEQ ID NO:12, (b) the amino acid sequence encoded by acDNA insert contained within plasmid pCRII-TMSP3 (ATCC Accession No.______), and (c) biologically active variants thereof.

[0008] Still another embodiment of the invention is a purifiedpolypeptide comprising an amino acid sequence selected from the groupconsisting of (a) the amino acid sequence shown in SEQ ID NO:12, (b) theamino acid sequence encoded by a cDNA insert contained within plasmidpCRII-TMSP3 (ATCC Accession No. ______), and (c) biologically activevariants thereof.

[0009] Even another embodiment of the invention is a fusion proteincomprising a polypeptide consisting of an amino acid sequence selectedfrom the group consisting of (a) the amino acid sequence shown in SEQ IDNO:12, (b) the amino acid sequence encoded by a cDNA insert containedwithin plasmid pCRII-TMSP3 (ATCC Accession No. ______), and (c)biologically active variants thereof.

[0010] Another embodiment of the invention is a method of producing apolypeptide comprising an amino acid sequence selected from the groupconsisting of (a) the amino acid sequence shown in SEQ ID NO:12, (b) theamino acid sequence encoded by a cDNA insert contained within plasmidpCRII-TMSP3 (ATCC Accession No. ______), and (c) biologically activevariants thereof. A host cell comprising an expression vector thatencodes the polypeptide is cultured under conditions whereby thepolypeptide is expressed. The polypeptide is isolated.

[0011] Yet another embodiment of the invention is a method of detectinga coding sequence for a polypeptide comprising an amino acid sequenceselected from the group consisting of (a) the amino acid sequence shownin SEQ ID NO:12, (b) the amino acid sequence encoded by a cDNA insertcontained within plasmid pCRII-TMSP3 (ATCC Accession No. ______), and(c) biologically active variants thereof. A polynucleotide comprising 11contiguous nucleotides selected from the group consisting of (a) thecomplement of the nucleotide sequence shown in SEQ ID NO:11, (b) thecomplement of the coding sequence of the cDNA insert of plasmidpCRII-TMSP3, (c) a polynucleotide that hybridizes under stringentconditions to (a) or (b), (d) a polynucleotide having a nucleic acidsequence that deviates from the nucleic acid sequences specified in (a)to (c) due to the degeneration of the genetic code, and (e) apolynucleotide that represents a fragment, derivative, or allelicvariation of a nucleic acid sequence specified in (a) to (d) ishybridized to nucleic acid material of a biological sample to form ahybridization complex. The hybridization complex is detected.

[0012] Even another embodiment of the invention is a kit for detecting acoding sequence for a polypeptide comprising an amino acid sequenceselected from the group consisting of (a) the amino acid sequence shownin SEQ ID NO:12, (b) the amino acid sequence encoded by a cDNA insertcontained within plasmid pCRII-TMSP3 (ATCC Accession No. ______), and(c) biologically active variants thereof. The kit comprises apolynucleotide and instructions for detecting the coding sequence. Thepolynucleotide comprises 11 contiguous nucleotides selected from thegroup consisting of (a) the complement of the nucleotide sequence shownin SEQ ID NO:11, (b) the complement of the coding sequence of the cDNAinsert of plasmid pCRII-TMSP3 to nucleic acid material of a biologicalsample to form a hybridization complex, (c) a polynucleotide thathybridizes under stringent conditions to (a) or (b), (d) apolynucleotide having a nucleic acid sequence that deviates from thenucleic acid sequences specified in (a) to (c) due to the degenerationof the genetic code, and (e) a polynucleotide that represents afragment, derivative, or allelic variation of a nucleic acid sequencespecified in (a) to (d).

[0013] Still another embodiment of the invention is a method ofdetecting a polypeptide comprising an amino acid sequence selected fromthe group consisting of (a) the amino acid sequence shown in SEQ IDNO:12, (b) the amino acid sequence encoded by a cDNA insert containedwithin plasmid pCRII-TMSP3 (ATCC Accession No. ______), and (c)biologically active variants thereof. A biological sample is contactedwith a reagent that specifically binds to the polypeptide to form areagent-polypeptide complex. The reagent-polypeptide complex isdetected.

[0014] Yet another embodiment of the invention is a kit for detecting apolypeptide comprising an amino acid sequence selected from the groupconsisting of (a) the amino acid sequence shown in SEQ ID NO:12, (b) theamino acid sequence encoded by a cDNA insert contained within plasmidpCRII-TMSP3 (ATCC Accession No. ______), and (c) biologically activevariants thereof. The kit comprises an antibody which specifically bindsto the polypeptide and instructions for detecting the polypeptide.

[0015] Even another embodiment of the invention is a method of screeningfor agents that can regulate an activity of a human transmembrane serineprotease. A test compound is contacted with a polypeptide comprising anamino acid sequence selected from the group consisting of: (a) the aminoacid sequence shown in SEQ ID NO:12, (b) the amino acid sequence encodedby a cDNA insert contained within plasmid pCRII-TMSP3 (ATCC AccessionNo. ______), and (c) biologically active variants thereof. Binding ofthe test compound to the polypeptide is detected. A test compound thatbinds to the polypeptide is thereby identified as a potential agent forregulating the activity of the human transmembrane serine protease.

[0016] A further embodiment of the invention is a method of screeningfor therapeutic agents that can regulate an enzymatic activity of ahuman transmembrane serine protease. A test compound is contacted with apolypeptide comprising an amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence shown in SEQ ID NO:12, (b)the amino acid sequence encoded by a cDNA insert contained withinplasmid pCRII-TMSP3 (ATCC Accession No. ______), and (c) biologicallyactive variants thereof. The enzymatic activity of the polypeptide isdetected. A test compound that increases the enzymatic activity of thepolypeptide is thereby identified as a potential therapeutic agent forincreasing the enzymatic activity of the human transmembrane serineprotease. A test compound that decreases the enzymatic activity of thepolypeptide is thereby identified as a potential therapeutic agent fordecreasing the enzymatic activity of the human transmembrane serineprotease.

[0017] Still another embodiment of the invention is a method ofscreening for therapeutic agents that can regulate an activity of ahuman transmembrane serine protease. A test compound is contacted with aproduct encoded by a polynucleotide comprising a nucleotide sequenceselected from the group consisting of (a) the amino acid sequence shownin SEQ ID NO:12, (b) the amino acid sequence encoded by a cDNA insertcontained within plasmid pCRII-TMSP3 (ATCC Accession No. ______), and(c) biologically active variants thereof. Binding of the test compoundto the product is detected. A test compound that binds to the product isthereby identified as a potential therapeutic agent for regulating theactivity of the human transmembrane serine protease.

[0018] Another embodiment of the invention is a method of reducing anactivity of a human transmembrane serine protease. A cell comprising thehuman transmembrane serine protease is contacted with a reagent thatspecifically binds to a product encoded by a polynucleotide comprising anucleotide sequence selected from the group consisting of (a) the aminoacid sequence shown in SEQ ID NO:12, (b) the amino acid sequence encodedby a cDNA insert contained within plasmid pCRII-TMSP3 (ATCC AccessionNo. ______), and (c) biologically active variants thereof. The activityof the human transmembrane serine protease is thereby reduced.

[0019] Yet another embodiment of the invention is a pharmaceuticalcomposition, comprising a reagent and a pharmaceutically acceptablecarrier. The reagent specifically binds to a polypeptide comprising anamino acid sequence selected from the group consisting of (a) the aminoacid sequence shown in SEQ ID NO:12, (b) the amino acid sequence encodedby a cDNA insert contained within plasmid pCRII-TMSP3 (ATCC AccessionNo. ______), and (c) biologically active variants thereof; and

[0020] Even another embodiment of the invention is a pharmaceuticalcomposition comprising a reagent and a pharmaceutically acceptablecarrier. The reagent specifically binds to a product of a polynucleotidecomprising a coding sequence selected from the group consisting of (a)the amino acid sequence shown in SEQ ID NO:12, (b) the amino acidsequence encoded by a cDNA insert contained within plasmid pCRII-TMSP3(ATCC Accession No. ______), and (c) biologically active variantsthereof.

[0021] A further embodiment of the invention is a pharmaceuticalcomposition comprising an expression vector and a pharmaceuticallyacceptable carrier. The expression vector encodes a polypeptidecomprising an amino acid sequence selected from the group consisting of(a) the amino acid sequence shown in SEQ ID NO:12, (b) the amino acidsequence encoded by a cDNA insert contained within plasmid pCRII-TMSP3(ATCC Accession No. ______), and (c) biologically active variantsthereof.

[0022] Still another embodiment of the invention is a method of treatinga disorder selected from the group consisting of chronic obstructivepulmonary disease, cancer, metastasis of malignant cells, tumorangiogenesis, inflammation, atherosclerosis, neurodegenerative diseases,and pathogenic infections. A therapeutically effective dose of a reagentthat inhibits a function of a human transmembrane serine protease isadministered to a patient in need thereof. The human transmembraneserine protease comprises an amino acid sequence selected from the groupconsisting of (a) the amino acid sequence shown in SEQ ID NO:12, (b) theamino acid sequence encoded by a cDNA insert contained within plasmidpCRII-TMSP3 (ATCC Accession No. ______), and (c) biologically activevariants thereof. Symptoms of the disorder are thereby ameliorated.

[0023] Even another embodiment of the invention is a isolatedpolynucleotide selected from the group consisting of: (a) apolynucleotide encoding a protein that comprises the amino acid sequenceof SEQ ID NO:12, (b) a polynucleotide comprising the sequence of SEQ IDNO:11, (c) a polynucleotide comprising a coding sequence of a cDNAcontained within plasmid pCRII-TMSP3 (ATCC Accession No. ______), (d) apolynucleotide encoding a protein that comprises the amino acid sequenceencoded by the cDNA of plasmid pCRII-TMSP3, (e) a polynucleotide whichhybridizes under stringent conditions to a polynucleotide specified in(a)-(d); (e) a polynucleotide having a nucleic acid sequence thatdeviates from the nucleic acid sequences specified in (a)-(d) due to thedegeneration of the genetic code, and (f) a polynucleotide thatrepresents a fragment, derivative, or allelic variation of a nucleicacid sequence specified in (a)-(e).

[0024] Yet another embodiment of the invention is an expression vectorcomprising polynucleotide selected from the group consisting of: (a) apolynucleotide encoding a protein that comprises the amino acid sequenceof SEQ ID NO:12, (b) a polynucleotide comprising the sequence of SEQ IDNO:11, (c) a polynucleotide comprising a coding sequence of a cDNAcontained within plasmid pCRII-TMSP3 (ATCC Accession No. ______), (d) apolynucleotide encoding a protein that comprises the amino acid sequenceencoded by the cDNA of plasmid pCRII-TMSP3, (e) a polynucleotide whichhybridizes under stringent conditions to a polynucleotide specified in(a)-(d); (e) a polynucleotide having a nucleic acid sequence thatdeviates from the nucleic acid sequences specified in (a)-(d) due to thedegeneration of the genetic code, and (f) a polynucleotide thatrepresents a fragment, derivative, or allelic variation of a nucleicacid sequence specified in (a)-(e).

[0025] A further embodiment of the invention is a host cell comprisingan expression vector comprising polynucleotide selected from the groupconsisting of: (a) a polynucleotide encoding a protein that comprisesthe amino acid sequence of SEQ ID NO:12, (b) a polynucleotide comprisingthe sequence of SEQ ID NO:11, (c) a polynucleotide comprising a codingsequence of a cDNA contained within plasmid pCRII-TMSP3 (ATCC AccessionNo. ______), (d) a polynucleotide encoding a protein that comprises theamino acid sequence encoded by the cDNA of plasmid pCRII-TMSP3, (e) apolynucleotide which hybridizes under stringent conditions to apolynucleotide specified in (a)-(d); (e) a polynucleotide having anucleic acid sequence that deviates from the nucleic acid sequencesspecified in (a)-(d) due to the degeneration of the genetic code, and(f) a polynucleotide that represents a fragment, derivative, or allelicvariation of a nucleic acid sequence specified in (a)-(e).

[0026] Another embodiment of the invention is a preparation ofantibodies that specifically bind to a polypeptide selected from thegroup consisting of (a) the amino acid sequence shown in SEQ ID NO:12,(b) the amino acid sequence encoded by a cDNA insert contained withinplasmid pCRII-TMSP3 (ATCC Accession No. ______), and (c) biologicallyactive variants thereof.

[0027] Even another embodiment of the invention is a antisenseoligonucleotide that hybridizes to a polynucleotide selected from thegroup consisting of (a) a polynucleotide encoding a protein thatcomprises the amino acid sequence of SEQ ID NO:12, (b) a polynucleotidecomprising the sequence of SEQ ID NO:11, (c) a polynucleotide comprisinga coding sequence of a cDNA contained within plasmid pCRII-TMSP3 (ATCCAccession No. ______), (d) a polynucleotide encoding a protein thatcomprises the amino acid sequence encoded by the cDNA of plasmidpCRII-TMSP3, (e) a polynucleotide which hybridizes under stringentconditions to a polynucleotide specified in (a)-(d); (e) apolynucleotide having a nucleic acid sequence that deviates from thenucleic acid sequences specified in (a)-(d) due to the degeneration ofthe genetic code, and (f) a polynucleotide that represents a fragment,derivative, or allelic variation of a nucleic acid sequence specified in(a)-(e).

[0028] The invention thus provides reagents and methods for regulatinghuman transmembrane serine protease activity, which can be used interalia, to treat COPD, metastasis of malignant cells, tumor angiogenesis,inflammation, atherosclerosis, neurodegenerative diseases, andpathogenic infections.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1. Alignment of human transmembrane serine protease as shownin SEQ ID NO:12 with the protein identified by SwissProt Accession No.O15393 (SEQ ID NO:14).

[0030]FIG. 2. Prosite search results.

[0031]FIG. 3. BLOCKS search results.

[0032]FIG. 4. Relative expression of human transmembrane serine proteasein respiratory cells and tissues.

[0033]FIG. 5. Relative expression of human transmembrane serine proteasein various human tissues and the neutrophil-like cell line HL60.

[0034]FIG. 6. Northern blot showing expression of human transmembraneserine protease.

[0035]FIG. 7. Relative expression of human transmembrane serine proteasein various tissues.

[0036]FIG. 8. Fold change in expression of human transmembrane serineprotease in various cancer cells.

[0037]FIG. 9. Restriction map of pCRII-TMSP3.

DETAILED DESCRIPTION OF THE INVENTION

[0038] It is a discovery of the present invention that regulators of ahuman transmembrane serine protease can be used to regulate degradationof the extracellular matrix. Human transmembrane serine protease asshown in SEQ ID NO:12 is 38% identical over a 370 amino acid overlap tothe protein identified by SwissProt Accession No. O15393 (SEQ ID NO:14)and annotated as a transmembrane serine protease 2 (FIG. 1). RelatedESTs (SEQ ID NOS:1-8) are expressed in placenta, breast, colon, andovarian tumor. The results of Prosite and BLOCKS searches are shown inFIGS. 2 and 3, respectively. Human transmembrane serine protease istherefore expected to be useful for the same purposes as previouslyidentified serine proteases.

[0039] Polypeptides

[0040] Transmembrane serine protease polypeptides according to theinvention comprise at least 10, 15, 25, 50, 75, 100, 125, 150, 175, 200,225, 250, 275, 300, 350, 400, or 410 contiguous amino acids selectedfrom SEQ ID NO:12 or from a biologically active variant thereof, asdefined below. A transmembrane serine protease polypeptide of theinvention therefore can be a portion of a transmembrane serine proteasemolecule, a full-length transmembrane serine protease molecule, or afusion protein comprising all or a portion of a transmembrane serineprotease molecule.

[0041] Biologically Active Variants

[0042] Transmembrane serine protease variants that are biologicallyactive, i.e., retain a transmembrane serine protease activity, also aretransmembrane serine protease polypeptides. Preferably, naturally ornon-naturally occurring transmembrane serine protease variants haveamino acid sequences which are at least about 50, preferably about 75,90, 96, or 98% identical to an amino acid sequence shown in SEQ IDNO:12. Percent identity between a putative transmembrane serine proteasevariant and an amino acid sequence of SEQ ID NO:12 is determined usingthe Blast2 alignment program (Blosum62, Expect 10, standard geneticcodes).

[0043] Variations in percent identity can be due, for example, to aminoacid substitutions, insertions, or deletions. Amino acid substitutionsare defined as one for one amino acid replacements. They areconservative in nature when the substituted amino acid has similarstructural and/or chemical properties. Examples of conservativereplacements are substitution of a leucine with an isoleucine or valine,an aspartate with a glutamate, or a threonine with a serine.

[0044] Amino acid insertions or deletions are changes to or within anamino acid sequence. They typically fall in the range of about 1 to 5amino acids. Guidance in determining which amino acid residues can besubstituted, inserted, or deleted without abolishing biological orimmunological activity can be found using computer programs well knownin the art, such as DNASTAR software. Whether an amino acid changeresults in a biologically active transmembrane serine proteasepolypeptide can readily be determined by assaying for fibronectinbinding or for transmembrane serine protease activity, as is known inthe art and described, for example, in Example 2.

[0045] Fusion Proteins

[0046] Fusion proteins are useful for generating antibodies againsttransmembrane serine protease amino acid sequences and for use invarious assay systems. For example, fusion proteins can be used toidentify proteins that interact with portions of a transmembrane serineprotease polypeptide, including its active site and fibronectin domains.Methods such as protein affinity chromatography or library-based assaysfor protein-protein interactions, such as the yeast two-hybrid or phagedisplay systems, can be used for this purpose. Such methods are wellknown in the art and also can be used as drug screens.

[0047] A transmembrane serine protease fusion protein comprises twoprotein segments fused together by means of a peptide bond. For example,the first protein segment can comprise at least 10, 15, 25, 50, 75, 100,125, 150, 175, 200, 225, 250, 275, 300, 350, 400, or 410 contiguousamino acids selected from SEQ ID NO:12 or a biologically active variantthereof. Preferably, a fusion protein comprises the active site of theprotease, one or both of the trypsin_ser or trypsin_his domains, or oneor more of the functional domains identified in FIGS. 1-3. The firstprotein segment also can comprise full-length transmembrane serineprotease.

[0048] The second protein segment can be a full-length protein or aprotein fragment or polypeptide. Proteins commonly used in fusionprotein construction include β-galactosidase, β-glucuronidase, greenfluorescent protein (GFP), autofluorescent proteins, including bluefluorescent protein (BFP), glutathione-S-transferase (GST), luciferase,horseradish peroxidase (HRP), and chloramphenicol acetyltransferase(CAT). Additionally, epitope tags are used in fusion proteinconstructions, including histidine (His) tags, FLAG tags, influenzahemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx)tags. Other fusion constructions can include maltose binding protein(MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA bindingdomain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Afusion protein also can be engineered to contain a cleavage site locatedbetween the transmembrane serine protease polypeptide-encoding sequenceand the heterologous protein sequence, so that the transmembrane serineprotease polypeptide can be cleaved and purified away from theheterologous moiety.

[0049] A fusion protein can be synthesized chemically, as is known inthe art. Preferably, a fusion protein is produced by covalently linkingtwo protein segments or by standard procedures in the art of molecularbiology. Recombinant DNA methods can be used to prepare fusion proteins,for example, by making a DNA construct which comprises transmembraneserine protease coding sequences disclosed herein in proper readingframe with nucleotides encoding the second protein segment andexpressing the DNA construct in a host cell, as is known in the art.Many kits for constructing fusion proteins are available from companiessuch as Promega Corporation (Madison, Wis.), Stratagene (La Jolla,Calif.), CLONTECH (Mountain View, Calif.), Santa Cruz Biotechnology(Santa Cruz, Calif.), MBL International Corporation (MIC; Watertown,Mass.), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).

[0050] Identification of Species Homologs

[0051] Species homologs of human transmembrane serine protease can beobtained using transmembrane serine protease polynucleotides (describedbelow) to make suitable probes or primers to screening cDNA expressionlibraries from other species, such as mice, monkeys, or yeast,identifying cDNAs which encode homologs of transmembrane serineprotease, and expressing the cDNAs as is known in the art.

[0052] Polynucleotides

[0053] A transmembrane serine protease polynucleotide can be single- ordouble-stranded and comprises a coding sequence or the complement of acoding sequence for a transmembrane serine protease polypeptide. Acoding sequence for the transmembrane serine protease of SEQ ID NO:12 isshown in SEQ ID NO:11.

[0054] Degenerate nucleotide sequences encoding human transmembraneserine protease polypeptides, as well as homologous nucleotide sequenceswhich are at least about 50, 55, 60, 65, 70, preferably about 75, 90,96, or 98% identical to the transmembrane serine protease codingsequence shown in SEQ ID NO:11 also are transmembrane serine proteasepolynucleotides. Percent sequence identity between the sequences of twopolynucleotides is determined using computer programs such as ALIGNwhich employ the FASTA algorithm, using an affine gap search with a gapopen penalty of −12 and a gap extension penalty of −2. Complementary DNA(cDNA) molecules, species homologs, and variants of transmembrane serineprotease polynucleotides that encode biologically active transmembraneserine protease polypeptides also are transmembrane serine proteasepolynucleotides.

[0055] Identification of Variants and Homologs

[0056] Variants and homologs of the transmembrane serine proteasepolynucleotides disclosed above also are transmembrane serine proteasepolynucleotides. Typically, homologous transmembrane serine proteasepolynucleotide sequences can be identified by hybridization of candidatepolynucleotides to known transmembrane serine protease polynucleotidesunder stringent conditions, as is known in the art. For example, usingthe following wash conditions—2×SSC (0.3 M NaCl, 0.03 M sodium citrate,pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2×SSC,0.1% SDS, 50° C. once, 30 minutes; then 2×SSC, room temperature twice,10 minutes each—homologous sequences can be identified which contain atmost about 25-30% basepair mismatches. More preferably, homologousnucleic acid strands contain 15-25% basepair mismatches, even morepreferably 5-15% basepair mismatches.

[0057] Species homologs of the transmembrane serine proteasepolynucleotides disclosed herein can be identified by making suitableprobes or primers and screening cDNA expression libraries from otherspecies, such as mice, monkeys, or yeast. Human variants oftransmembrane serine protease polynucleotides can be identified, forexample, by screening human cDNA expression libraries. It is well knownthat the T_(m) of a double-stranded DNA decreases by 1-1.5° C. withevery 1% decrease in homology (Bonner et al., J. Mol. Biol. 81, 123(1973). Variants of human transmembrane serine protease polynucleotidesor transmembrane serine protease polynucleotides of other species cantherefore be identified, for example, by hybridizing a putativehomologous transmembrane serine protease polynucleotide with apolynucleotide having a nucleotide sequence of SEQ ID NO:11 to form atest hybrid. The melting temperature of the test hybrid is compared withthe melting temperature of a hybrid comprising transmembrane serineprotease polynucleotides having perfectly complementary nucleotidesequences, and the number or percent of basepair mismatches within thetest hybrid is calculated.

[0058] Nucleotide sequences which hybridize to transmembrane serineprotease polynucleotides or their complements following stringenthybridization and/or wash conditions are also transmembrane serineprotease polynucleotides. Stringent wash conditions are well known andunderstood in the art and are disclosed, for example, in Sambrook etal., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages9.50-9.51.

[0059] Typically, for stringent hybridization conditions a combinationof temperature and salt concentration should be chosen that isapproximately 12-20° C. below the calculated T_(m) of the hybrid understudy. The T_(m) of a hybrid between a transmembrane serine proteasepolynucleotide having a coding sequence disclosed herein and apolynucleotide sequence which is at least about 50, 55, 60, 65, 70,preferably about 75, 90, 96, or 98% identical to that nucleotidesequence can be calculated, for example, using the equation of Boltonand McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):

T _(m)=81.5° C.−16.6(log₁₀ [Na⁺])+0.41(% G+C)−0.63(% formamide)−600/l),

[0060] where l=the length of the hybrid in basepairs.

[0061] Stringent wash conditions include, for example, 4×SSC at 65° C.,or 50% formamide, 4×SSC at 42° C., or 0.5×SSC, 0.1% SDS at 65° C. Highlystringent wash conditions include, for example, 0.2×SSC at 65° C.

[0062] Preparation of Polynucleotides

[0063] A naturally occurring transmembrane serine proteasepolynucleotide can be isolated free of other cellular components such asmembrane components, proteins, and lipids. Polynucleotides can be madeby a cell and isolated using standard nucleic acid purificationtechniques, synthesized using an amplification technique, such as thepolymerase chain reaction (PCR), or synthesized using an automaticsynthesizer. Methods for isolating polynucleotides are routine and areknown in the art. Any such technique for obtaining a polynucleotide canbe used to obtain isolated transmembrane serine proteasepolynucleotides. For example, restriction enzymes and probes can be usedto isolate polynucleotide fragments that comprise transmembrane serineprotease nucleotide sequences. Isolated polynucleotides are inpreparations that are free or at least 70, 80, or 90% free of othermolecules.

[0064] Transmembrane serine protease cDNA molecules can be made withstandard molecular biology techniques, using transmembrane serineprotease mRNA as a template. Transmembrane serine protease cDNAmolecules can thereafter be replicated using molecular biologytechniques known in the art and disclosed in manuals such as Sambrook etal. (1989). An amplification technique, such as PCR, can be used toobtain additional copies of transmembrane serine proteasepolynucleotides, using either human genomic DNA or cDNA as a template.

[0065] Alternatively, synthetic chemistry techniques can be used tosynthesize transmembrane serine protease polynucleotides. The degeneracyof the genetic code allows alternate nucleotide sequences to besynthesized which will encode a transmembrane serine proteasepolypeptide having, for example, the amino acid sequence shown in SEQ IDNO:12 or a biologically active variant of that sequence.

[0066] Extending Polynucleotides

[0067] Various PCR-based methods can be used to extend the nucleic acidsequences encoding the disclosed portions of human transmembrane serineprotease to detect upstream sequences such as promoters and regulatoryelements. For example, restriction-site PCR uses universal primers toretrieve unknown sequence adjacent to a known locus (Sarkar, PCR MethodsApplic. 2, 318-322, 1993). Genomic DNA is first amplified in thepresence of a primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

[0068] Inverse PCR also can be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia et al., Nucleic AcidsRes. 16, 8186, 1988). Primers can be designed using commerciallyavailable software, such as OLIGO 4.06 Primer Analysis software(National Biosciences Inc., Plymouth, Minn.), to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

[0069] Another method which can be used is capture PCR, which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom et al., PCR MethodsApplic. 1, 111-119, 1991). In this method, multiple restriction enzymedigestions and ligations are used to place an engineered double-strandedsequence into an unknown fragment of the DNA molecule before performingPCR.

[0070] Another method that can be used to retrieve unknown sequences isthat of Parker et al., Nucleic Acids Res. 19, 3055-3060, 1991.Additionally, PCR, nested primers, and PROMOTERFINDER libraries(CLONTECH, Palo Alto, Calif.) can be used to walk genomic DNA. Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

[0071] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences that contain the 5′ regions of genes. Use of a randomly primedlibrary may be especially preferable for situations in which an oligod(T) library does not yield a full-length cDNA. Genomic libraries can beuseful for extension of sequence into 5′ non-transcribed regulatoryregions.

[0072] Commercially available capillary electrophoresis systems can beused to analyze the size or confirm the nucleotide sequence of PCR orsequencing products. For example, capillary sequencing can employflowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devicecamera. Output/light intensity can be converted to electrical signalusing appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR,Perkin Elmer), and the entire process from loading of samples tocomputer analysis and electronic data display can be computercontrolled. Capillary electrophoresis is especially preferable for thesequencing of small pieces of DNA that might be present in limitedamounts in a particular sample.

[0073] Obtaining Polypeptides

[0074] Transmembrane serine protease polypeptides can be obtained, forexample, by purification from cells, by expression of transmembraneserine protease polynucleotides, or by direct chemical synthesis.

[0075] Protein Purification

[0076] Transmembrane serine protease polypeptides can be purified fromcells, such as primary tumor cells, metastatic cells, or cancer celllines (e.g., colon cancer cell lines HCT116, DLD1, HT29, Caco2, SW837,SW480, and RKO, breast cancer cell lines 21-PT, 21-MT, MDA-468, SK-BR3,and BT-474, the A549 lung cancer cell line, or the H392 glioblastomacell line), as well as cells transfected with a transmembrane serineprotease expression construct. Placenta, breast, colon, and ovariantumor are especially useful sources of transmembrane serine proteasepolypeptides. A purified transmembrane serine protease polypeptide isseparated from other compounds that normally associate with thetransmembrane serine protease polypeptide in the cell, such as certainproteins, carbohydrates, or lipids, using methods well-known in the art.Such methods include, but are not limited to, size exclusionchromatography, ammonium sulfate fractionation, ion exchangechromatography, affinity chromatography, and preparative gelelectrophoresis. A preparation of purified transmembrane serine proteasepolypeptides is at least 80% pure; preferably, the preparations are 90%,95%, or 99% pure. Purity of the preparations can be assessed by anymeans known in the art, such as SDS-polyacrylamide gel electrophoresis.Enzymatic activity of the purified preparations can be assayed, forexample, as described in Example 2.

[0077] Expression of Polynucleotides

[0078] To express a transmembrane serine protease polypeptide, atransmembrane serine protease polynucleotide can be inserted into anexpression vector which contains the necessary elements for thetranscription and translation of the inserted coding sequence. Methodsthat are well known to those skilled in the art can be used to constructexpression vectors containing sequences encoding transmembrane serineprotease polypeptides and appropriate transcriptional and translationalcontrol elements. These methods include in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.Such techniques are described, for example, in Sambrook et al. (1989)and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons, New York, N.Y, 1989.

[0079] A variety of expression vector/host systems can be utilized tocontain and express sequences encoding a transmembrane serine proteasepolypeptide. These include, but are not limited to, microorganisms, suchas bacteria transformed with recombinant bacteriophage, plasmid, orcosmid DNA expression vectors; yeast transformed with yeast expressionvectors, insect cell systems infected with virus expression vectors(e.g., baculovirus), plant cell systems transformed with virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322plasmids), or animal cell systems.

[0080] The control elements or regulatory sequences are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements can vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, can be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or pSPORT1 plasmid (Life Technologies) and the like canbe used. The baculovirus polyhedrin promoter can be used in insectcells. Promoters or enhancers derived from the genomes of plant cells(e.g., heat shock, RUBISCO, and storage protein genes) or from plantviruses (e.g., viral promoters or leader sequences) can be cloned intothe vector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of a nucleotide sequenceencoding a transmembrane serine protease polypeptide, vectors based onSV40 or EBV can be used with an appropriate selectable marker.

[0081] Bacterial and Yeast Expression Systems

[0082] In bacterial systems, a number of expression vectors can beselected depending upon the use intended for the transmembrane serineprotease polypeptide. For example, when a large quantity of atransmembrane serine protease polypeptide is needed for the induction ofantibodies, vectors which direct high level expression of fusionproteins that are readily purified can be used. Such vectors include,but are not limited to, multifunctional E. coli cloning and expressionvectors such as BLUESCRIPT (Stratagene), in which the sequence encodingthe transmembrane serine protease polypeptide can be ligated into thevector in frame with sequences for the amino-terminal Met and thesubsequent 7 residues of β-galactosidase so that a hybrid protein isproduced. pIN vectors (Van Heeke & Schuster, J. Biol. Chem. 264,5503-5509, 1989 or pGEX vectors (Promega, Madison, Wis.) can be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems can be designed to includeheparin, thrombin, or Factor Xa protease cleavage sites so that thecloned polypeptide of interest can be released from the GST moiety atwill.

[0083] In the yeast Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH can be used. For reviews, see Ausubel et al.(1989) and Grant et al., Methods Enzymol. 153, 516-544, 1987.

[0084] Plant and Insect Expression Systems

[0085] If plant expression vectors are used, the expression of sequencesencoding transmembrane serine protease polypeptides can be driven by anyof a number of promoters. For example, viral promoters such as the 35Sand 19S promoters of CaMV can be used alone or in combination with theomega leader sequence from TMV (Takamatsu EMBO J. 6, 307-311, 1987).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters can be used (Coruzzi et al., EMBO J. 3, 1671-1680,1984; Broglie et al., Science 224, 838-843, 1984; Winter et al., ResultsProbl. Cell Differ. 17, 85-105, 1991). These constructs can beintroduced into plant cells by direct DNA transformation or bypathogen-mediated transfection. Such techniques are described in anumber of generally available reviews (see, for example, Hobbs orMurray, in MCGRAW HILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill,New York, N.Y., pp. 191-196, 1992).

[0086] An insect system also can be used to express a transmembraneserine protease polypeptide. For example, in one such system Autographacalifornica nuclear polyhedrosis virus (AcNPV) is used as a vector toexpress foreign genes in Spodoptera frugiperda cells or in Trichoplusialarvae. Sequences encoding transmembrane serine protease polypeptidescan be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of transmembrane serine protease polypeptides willrender the polyhedrin gene inactive and produce recombinant viruslacking coat protein. The recombinant viruses can then be used toinfect, for example, S. frugiperda cells or Trichoplusia larvae in whichtransmembrane serine protease polypeptides can be expressed (Engelhardet al., Proc. Nat. Acad. Sci. 91, 3224-3227, 1994).

[0087] Mammalian Expression Systems

[0088] A number of viral-based expression systems can be utilized inmammalian host cells. For example, if an adenovirus is used as anexpression vector, sequences encoding transmembrane serine proteasepolypeptides can be ligated into an adenovirus transcription/translationcomplex consisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome can beused to obtain a viable virus which is capable of expressing atransmembrane serine protease polypeptide in infected host cells (Logan& Shenk, Proc. Natl. Acad. Sci. 81, 3655-3659, 1984). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,can be used to increase expression in mammalian host cells.

[0089] Human artificial chromosomes (HACs) also can be used to deliverlarger fragments of DNA than can be contained and expressed in aplasmid. HACs of 6M to 10M are constructed and delivered to cells viaconventional delivery methods (e.g., liposomes, polycationic aminopolymers, or vesicles).

[0090] Specific initiation signals also can be used to achieve moreefficient translation of sequences encoding transmembrane serineprotease polypeptides. Such signals include the ATG initiation codon andadjacent sequences. In cases where sequences encoding a transmembraneserine protease polypeptide, its initiation codon, and upstreamsequences are inserted into the appropriate expression vector, noadditional transcriptional or translational control signals may beneeded. However, in cases where only coding sequence, or a fragmentthereof, is inserted, exogenous translational control signals (includingthe ATG initiation codon) should be provided. The initiation codonshould be in the correct reading frame to ensure translation of theentire insert. Exogenous translational elements and initiation codonscan be of various origins, both natural and synthetic. The efficiency ofexpression can be enhanced by the inclusion of enhancers that areappropriate for the particular cell system that is used (see Scharf etal., Results Probl. Cell Differ. 20, 125-162, 1994).

[0091] Host Cells

[0092] A host cell strain can be chosen for its ability to modulate theexpression of the inserted sequences or to process an expressedtransmembrane serine protease polypeptide in the desired fashion. Suchmodifications of the polypeptide include, but are not limited to,acetylation, carboxylation, glycosylation, phosphorylation, lipidation,and acylation. Post-translational processing which cleaves a “prepro”form of the polypeptide also can be used to facilitate correctinsertion, folding and/or function. Different host cells that havespecific cellular machinery and characteristic mechanisms forpost-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38),are available from the American Type Culture Collection (ATCC; 10801University Boulevard, Manassas, Va. 20110-2209) and can be chosen toensure the correct modification and processing of the foreign protein.

[0093] Stable expression is preferred for long-term, high-yieldproduction of recombinant proteins. For example, cell lines that stablyexpress transmembrane serine protease polypeptides can be transformedusing expression vectors that can contain viral origins of replicationand/or endogenous expression elements and a selectable marker gene onthe same or on a separate vector. Following the introduction of thevector, cells can be allowed to grow for 1-2 days in an enriched mediumbefore they are switched to a selective medium. The purpose of theselectable marker is to confer resistance to selection, and its presenceallows growth and recovery of cells that successfully express theintroduced transmembrane serine protease sequences. Resistant clones ofstably transformed cells can be proliferated using tissue culturetechniques appropriate to the cell type.

[0094] Any number of selection systems can be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler et al., Cell 11, 223-32,1977) and adenine phosphoribosyltransferase (Lowy et al., Cell 22,817-23, 1980). Genes that can be employed in tk⁻ or aprt⁻ cells,respectively. Also, antimetabolite, antibiotic, or herbicide resistancecan be used as the basis for selection. For example, dhfr confersresistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. 77,3567-70, 1980); npt confers resistance to the aminoglycosides, neomycinand G-418 (Colbere-Garapin et al., J. Mol. Biol. 150, 1-14, 1981); andals and pat confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murray, 1992 supra). Additionalselectable genes have been described, for example trpB, which allowscells to utilize indole in place of tryptophan, or hisD, which allowscells to utilize histinol in place of histidine (Hartman & Mulligan,Proc. Natl. Acad. Sci. 85, 8047-51, 1988). Visible markers such asanthocyanins, β-glucuronidase and its substrate GUS, and luciferase andits substrate luciferin, can be used to identify transformants and toquantify the amount of transient or stable protein expressionattributable to a specific vector system (Rhodes et al., Methods Mol.Biol. 55, 121-131, 1995).

[0095] Detecting Expression of Polypeptides

[0096] Although the presence of marker gene expression suggests that thetransmembrane serine protease polynucleotide is also present, itspresence and expression may need to be confirmed. For example, if asequence encoding a transmembrane serine protease polypeptide isinserted within a marker gene sequence, transformed cells containingsequences that encode a transmembrane serine protease polypeptide can beidentified by the absence of marker gene function. Alternatively, amarker gene can be placed in tandem with a sequence encoding atransmembrane serine protease polypeptide under the control of a singlepromoter. Expression of the marker gene in response to induction orselection usually indicates expression of the transmembrane serineprotease polynucleotide.

[0097] Alternatively, host cells which contain a transmembrane serineprotease polynucleotide and which express a transmembrane serineprotease polypeptide can be identified by a variety of procedures knownto those of skill in the art. These procedures include, but are notlimited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay orimmunoassay techniques that include membrane, solution, or chip-basedtechnologies for the detection and/or quantification of nucleic acid orprotein.

[0098] The presence of a polynucleotide sequence encoding atransmembrane serine protease polypeptide can be detected by DNA-DNA orDNA-RNA hybridization or amplification using probes or fragments orfragments of polynucleotides encoding a transmembrane serine proteasepolypeptide. Nucleic acid amplification-based assays involve the use ofoligonucleotides selected from sequences encoding a transmembrane serineprotease polypeptide to detect transformants that contain atransmembrane serine protease polynucleotide.

[0099] A variety of protocols for detecting and measuring the expressionof a transmembrane serine protease polypeptide, using either polyclonalor monoclonal antibodies specific for the polypeptide, are known in theart. Examples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).A two-site, monoclonal-based immunoassay using monoclonal antibodiesreactive to two non-interfering epitopes on a transmembrane serineprotease polypeptide can be used, or a competitive binding assay can beemployed. These and other assays are described in Hampton et al.,SEROLOGICAL METHODS: A LABORATORY MANUAL, APS Press, St. Paul, Minn.,1990) and Maddox et al., J. Exp. Med. 158, 1211-1216, 1983).

[0100] A wide variety of labels and conjugation techniques are known bythose skilled in the art and can be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encodingtransmembrane serine protease polypeptides include oligolabeling, nicktranslation, end-labeling, or PCR amplification using a labelednucleotide. Alternatively, sequences encoding a transmembrane serineprotease polypeptide can be cloned into a vector for the production ofan mRNA probe. Such vectors are known in the art, are commerciallyavailable, and can be used to synthesize RNA probes in vitro by additionof labeled nucleotides and an appropriate RNA polymerase, such as T7,T3, or SP6. These procedures can be conducted using a variety ofcommercially available kits (Amersham Pharmacia Biotech, Promega, and USBiochemical). Suitable reporter molecules or labels which can be usedfor ease of detection include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents, as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

[0101] Expression and Purification of Polypeptides

[0102] Host cells transformed with nucleotide sequences encoding atransmembrane serine protease polypeptide can be cultured underconditions suitable for the expression and recovery of the protein fromcell culture. The polypeptide produced by a transformed cell can besecreted or contained intracellularly depending on the sequence and/orthe vector used. As will be understood by those of skill in the art,expression vectors containing polynucleotides that encode transmembraneserine protease polypeptides can be designed to contain signal sequencesthat direct secretion of transmembrane serine protease polypeptidesthrough a prokaryotic or eukaryotic cell membrane.

[0103] Other constructions can be used to join a sequence encoding atransmembrane serine protease polypeptide to a nucleotide sequenceencoding a polypeptide domain that will facilitate purification ofsoluble proteins. Such purification facilitating domains include, butare not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor Xa orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and the transmembrane serine protease polypeptide can be used tofacilitate purification. One such expression vector provides forexpression of a fusion protein containing a transmembrane serineprotease polypeptide and 6 histidine residues preceding a thioredoxin oran enterokinase cleavage site. The histidine residues facilitatepurification on IMAC (immobilized metal ion affinity chromatography asdescribed in Porath et al., Prot. Exp. Purif. 3, 263-281, 1992), whilethe enterokinase cleavage site provides a means for purifying thetransmembrane serine protease polypeptide from the fusion protein.Vectors which contain fusion proteins are disclosed in Kroll et al., DNACell Biol. 12, 441-453, 1993).

[0104] Chemical Synthesis

[0105] Sequences encoding a transmembrane serine protease polypeptidecan be synthesized, in whole or in part, using chemical methods wellknown in the art (see Caruthers et al., Nucl. Acids Res. Symp. Ser.215-223, 1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225-232, 1980).Alternatively, a transmembrane serine protease polypeptide itself can beproduced using chemical methods to synthesize its amino acid sequence.For example, transmembrane serine protease polypeptides can be producedby direct peptide synthesis using solid-phase techniques (Merrifield, J.Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al., Science 269,202-204, 1995). Protein synthesis can be performed using manualtechniques or by automation. Automated synthesis can be achieved, forexample, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Various fragments of transmembrane serine protease polypeptidescan be separately synthesized and combined using chemical methods toproduce a full-length molecule.

[0106] The newly synthesized peptide can be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton,PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., NewYork, N.Y., 1983). The composition of a synthetic transmembrane serineprotease polypeptide can be confirmed by amino acid analysis orsequencing (e.g., the Edman degradation procedure; see Creighton,supra). Additionally, any portion of the amino acid sequence of thetransmembrane serine protease polypeptide can be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins to produce a variant polypeptide or a fusion protein.

[0107] Production of Altered Polypeptides

[0108] As will be understood by those of skill in the art, it may beadvantageous to produce transmembrane serine proteasepolypeptide-encoding nucleotide sequences possessing non-naturallyoccurring codons. For example, codons preferred by a particularprokaryotic or eukaryotic host can be selected to increase the rate ofprotein expression or to produce an RNA transcript having desirableproperties, such as a half-life that is longer than that of a transcriptgenerated from the naturally occurring sequence.

[0109] The nucleotide sequences disclosed herein can be engineered usingmethods generally known in the art to alter transmembrane serineprotease polypeptide-encoding sequences for a variety of reasons,including modification of the cloning, processing, and/or expression ofthe gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides can be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis can be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

[0110] Antibodies

[0111] Any type of antibody known in the art can be generated to bindspecifically to an epitope of a transmembrane serine proteasepolypeptide. “Antibody” as used herein includes intact immunoglobulinmolecules, as well as fragments thereof, such as Fab, F(ab′)₂, and Fv,which are capable of binding an epitope of a transmembrane serineprotease polypeptide. Typically, at least 6, 8, 10, or 12 contiguousamino acids are required to form an epitope. However, epitopes whichinvolve non-contiguous amino acids may require more, e.g., at least 15,25, or 50 amino acids.

[0112] An antibody which specifically binds to an epitope of atransmembrane serine protease polypeptide can be used therapeutically,as well as in immunochemical assays, including but not limited toWestern blots, ELISAs, radioimmunoassays, immunohistochemical assays,immunoprecipitations, or other immunochemical assays known in the art.Various immunoassays can be used to identify antibodies having thedesired specificity. Numerous protocols for competitive binding orimmunoradiometric assays are well known in the art. Such immunoassaystypically involve the measurement of complex formation between animmunogen and an antibody that specifically binds to the immunogen.

[0113] Typically, an antibody that specifically binds to a transmembraneserine protease polypeptide provides a detection signal at least 5-,10-, or 20-fold higher than a detection signal provided with otherproteins when used in an immunochemical assay. Preferably, antibodiesthat specifically bind to transmembrane serine protease polypeptides donot detect other proteins in immunochemical assays and canimmunoprecipitate a transmembrane serine protease polypeptide fromsolution.

[0114] Transmembrane serine protease polypeptides can be used toimmunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, orhuman, to produce polyclonal antibodies. If desired, a transmembraneserine protease polypeptide can be conjugated to a carrier protein, suchas bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.Depending on the host species, various adjuvants can be used to increasethe immunological response. Such adjuvants include, but are not limitedto, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), andsurface active substances (e.g. lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol). Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially useful.

[0115] Monoclonal antibodies that specifically bind to a transmembraneserine protease polypeptide can be prepared using any technique thatprovides for the production of antibody molecules by continuous celllines in culture. These techniques include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler et al., Nature 256, 495-497, 1985;Kozbor et al., J. Immunol. Methods 81, 31-42, 1985; Cote et al., Proc.Natl. Acad. Sci. 80, 2026-2030, 1983; Cole et al., Mol. Cell Biol. 62,109-120, 1984).

[0116] In addition, techniques developed for the production of “chimericantibodies,” the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity, can be used (Morrison et al., Proc. Natl. Acad.Sci. 81, 6851-6855, 1984; Neuberger et al., Nature 312, 604-608, 1984;Takeda et al., Nature 314, 452-454, 1985). Monoclonal and otherantibodies also can be “humanized” to prevent a patient from mounting animmune response against the antibody when it is used therapeutically.Such antibodies may be sufficiently similar in sequence to humanantibodies to be used directly in therapy or may require alteration of afew key residues. Sequence differences between rodent antibodies andhuman sequences can be minimized by replacing residues which differ fromthose in the human sequences by site directed mutagenesis of individualresidues or by grating of entire complementarity determining regions.Alternatively, one can produce humanized antibodies using recombinantmethods, as described in GB2188638B. Antibodies that specifically bindto a transmembrane serine protease polypeptide can contain antigenbinding sites which are either partially or fully humanized, asdisclosed in U.S. Pat. No. 5,565,332.

[0117] Alternatively, techniques described for the production of singlechain antibodies can be adapted using methods known in the art toproduce single chain antibodies that specifically bind to transmembraneserine protease polypeptides. Antibodies with related specificity, butof distinct idiotypic composition, can be generated by chain shufflingfrom random combinatorial immunoglobin libraries (Burton, Proc. Natl.Acad. Sci. 88, 11120-23, 1991).

[0118] Single-chain antibodies also can be constructed using a DNAamplification method, such as PCR, using hybridoma cDNA as a template(Thirion et al., 1996, Eur. J. Cancer Prev. 5, 507-11). Single-chainantibodies can be mono- or bispecific, and can be bivalent ortetravalent. Construction of tetravalent, bispecific single-chainantibodies is taught, for example, in Coloma & Morrison, 1997, Nat.Biotechnol. 15, 159-63. Construction of bivalent, bispecificsingle-chain antibodies is taught in Mallender & Voss, 1994, J. Biol.Chem. 269, 199-206.

[0119] A nucleotide sequence encoding a single-chain antibody can beconstructed using manual or automated nucleotide synthesis, cloned intoan expression construct using standard recombinant DNA methods, andintroduced into a cell to express the coding sequence, as describedbelow. Alternatively, single-chain antibodies can be produced directlyusing, for example, filamentous phage technology. Verhaar et al., 1995,Int. J. Cancer 61, 497-501; Nicholls et al., 1993, J. Immunol. Meth.165, 81-91.

[0120] Antibodies which specifically bind to transmembrane serineprotease polypeptides also can be produced by inducing in vivoproduction in the lymphocyte population or by screening immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi et al., Proc. Natl. Acad. Sci. 86, 3833-3837,1989; Winter et al., Nature 349, 293-299, 1991).

[0121] Other types of antibodies can be constructed and usedtherapeutically in methods of the invention. For example, chimericantibodies can be constructed as disclosed in WO 93/03151. Bindingproteins which are derived from immunoglobulins and which aremultivalent and multispecific, such as the “diabodies” described in WO94/13804, also can be prepared.

[0122] Antibodies of the invention can be purified by methods well knownin the art. For example, antibodies can be affinity purified by passageover a column to which a transmembrane serine protease polypeptide isbound. The bound antibodies can then be eluted from the column using abuffer with a high salt concentration.

[0123] Antisense Oligonucleotides

[0124] Antisense oligonucleotides are nucleotide sequences that arecomplementary to a specific DNA or RNA sequence. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form complexes and block either transcription ortranslation. Preferably, an antisense oligonucleotide is at least 11nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40,45, or 50 or more nucleotides long. Longer sequences also can be used.Antisense oligonucleotide molecules can be provided in a DNA constructand introduced into a cell as described above to decrease the level oftransmembrane serine protease gene products in the cell.

[0125] Antisense oligonucleotides can be deoxyribonucleotides,ribonucleotides, or a combination of both. Oligonucleotides can besynthesized manually or by an automated synthesizer, by covalentlylinking the 5′ end of one nucleotide with the 3′ end of anothernucleotide with non-phosphodiester internucleotide linkages suchalkylphosphonates, phosphorothioates, phosphorodithioates,alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphateesters, carbamates, acetamidate, carboxymethyl esters, carbonates, andphosphate triesters. See Brown, Meth. Mol. Biol. 20, 1-8, 1994;Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al., Chem. Rev.90, 543-583, 1990.

[0126] Modifications of transmembrane serine protease gene expressioncan be obtained by designing antisense oligonucleotides that will formduplexes to the control, 5′, or regulatory regions of the transmembraneserine protease gene. Oligonucleotides derived from the transcriptioninitiation site, e.g., between positions −10 and +10 from the startsite, are preferred. Similarly, inhibition can be achieved using “triplehelix” base-pairing methodology. Triple helix pairing is useful becauseit causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orchaperons. Therapeutic advances using triplex DNA have been described inthe literature (e.g., Gee et al., in Huber & Carr, MOLECULAR ANDIMMUNOLOGIC APPROACHES, Futura Publishing Co., Mt. Kisco, N.Y., 1994).An antisense oligonucleotide also can be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

[0127] Precise complementarity is not required for successful duplexformation between an antisense oligonucleotide and the complementarysequence of a transmembrane serine protease polynucleotide. Antisenseoligonucleotides which comprise, for example, 2, 3, 4, or 5 or morestretches of contiguous nucleotides which are precisely complementary toa transmembrane serine protease polynucleotide, each separated by astretch of contiguous nucleotides which are not complementary toadjacent transmembrane serine protease nucleotides, can providetargeting specificity for transmembrane serine protease mRNA.Preferably, each stretch of complementary contiguous nucleotides is atleast 4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementaryintervening sequences are preferably 1, 2, 3, or 4 nucleotides inlength. One skilled in the art can easily use the calculated meltingpoint of an antisense-sense pair to determine the degree of mismatchingwhich will be tolerated between a particular antisense oligonucleotideand a particular transmembrane serine protease polynucleotide sequence.

[0128] Antisense oligonucleotides can be modified without affectingtheir ability to hybridize to a transmembrane serine proteasepolynucleotide. These modifications can be internal or at one or bothends of the antisense molecule. For example, internucleoside phosphatelinkages can be modified by adding cholesteryl or diamine moieties withvarying numbers of carbon residues between the amino groups and terminalribose. Modified bases and/or sugars, such as arabinose instead ofribose, or a 3′, 5′-substituted oligonucleotide in which the 3′ hydroxylgroup or the 5′ phosphate group are substituted, also can be employed ina modified antisense oligonucleotide. These modified oligonucleotidescan be prepared by methods well known in the art. See, e.g., Agrawal etal., Trends Biotechnol. 10, 152-158, 1992; Uhlmann et al., Chem. Rev.90, 543-584, 1990; Uhlmann et al., Tetrahedron. Lett. 215, 3539-3542,1987.

[0129] Ribozymes

[0130] Ribozymes are RNA molecules with catalytic activity. See, e.g.,Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59,543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture& Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used toinhibit gene function by cleaving an RNA sequence, as is known in theart (e.g., Haseloff et al., U.S. Pat. No. 5,641,673). The mechanism ofribozyme action involves sequence-specific hybridization of the ribozymemolecule to complementary target RNA,-followed by endonucleolyticcleavage. Examples include engineered hammerhead motif ribozymemolecules that can specifically and efficiently catalyze endonucleolyticcleavage of specific nucleotide sequences.

[0131] The coding sequence of a transmembrane serine proteasepolynucleotide can be used to generate ribozymes which will specificallybind to mRNA transcribed from the transmembrane serine proteasepolynucleotide. Methods of designing and constructing ribozymes whichcan cleave other RNA molecules in trans in a highly sequence specificmanner have been developed and described in the art (see Haseloff et al.Nature 334, 585-591, 1988). For example, the cleavage activity ofribozymes can be targeted to specific RNAs by engineering a discrete“hybridization” region into the ribozyme. The hybridization regioncontains a sequence complementary to the target RNA and thusspecifically hybridizes with the target (see, for example, Gerlach etal., EP 321,201).

[0132] Specific ribozyme cleavage sites within a transmembrane serineprotease RNA target are initially identified by scanning the RNAmolecule for ribozyme cleavage sites which include the followingsequences: GUA, GUU, and GUC. Once identified, short RNA sequences ofbetween 15 and 20 ribonucleotides corresponding to the region of thetransmembrane serine protease target RNA containing the cleavage sitecan be evaluated for secondary structural features which may render thetarget inoperable. The suitability of candidate targets also can beevaluated by testing accessibility to hybridization with complementaryoligonucleotides using ribonuclease protection assays. Longercomplementary sequences can be used to increase the affinity of thehybridization sequence for the target. The hybridizing and cleavageregions of the ribozyme can be integrally related; thus, uponhybridizing to the transmembrane serine protease target RNA through thecomplementary regions, the catalytic region of the ribozyme can cleavethe target.

[0133] Ribozymes can be introduced into cells as part of a DNAconstruct. Mechanical methods, such as microinjection, liposome-mediatedtransfection, electroporation, or calcium phosphate precipitation, canbe used to introduce a ribozyme-containing DNA construct into cells inwhich it is desired to decrease transmembrane serine proteaseexpression. Alternatively, if it is desired that the cells stably retainthe DNA construct, it can be supplied on a plasmid and maintained as aseparate element or integrated into the genome of the cells, as is knownin the art. The DNA construct can include transcriptional regulatoryelements, such as a promoter element, an enhancer or UAS element, and atranscriptional terminator signal, for controlling transcription ofribozymes in the cells.

[0134] As taught in Haseloff et al., U.S. Pat. No. 5,641,673, ribozymescan be engineered so that ribozyme expression will occur in response tofactors that induce expression of a target gene. Ribozymes also can beengineered to provide an additional level of regulation, so thatdestruction of transmembrane serine protease mRNA occurs only when botha ribozyme and a target gene are induced in the cells.

[0135] Differentially Expressed Genes

[0136] Described herein are methods for the identification of geneswhose products interact with human transmembrane serine protease. Suchgenes may represent genes that are differentially expressed in disordersincluding, but not limited to, COPD, CNS disorders, cardiovasculardisorders, and cancer. Further, such genes may represent genes that aredifferentially regulated in response to manipulations relevant to theprogression or treatment of such diseases. Additionally, such genes mayhave a temporally modulated expression, increased or decreased atdifferent stages of tissue or organism development. A differentiallyexpressed gene may also have its expression modulated under controlversus experimental conditions. In addition, the human transmembraneserine protease gene or gene product may itself be tested fordifferential expression.

[0137] The degree to which expression differs in a normal versus adiseased state need only be large enough to be visualized via standardcharacterization techniques such as differential display techniques.Other such standard characterization techniques by which expressiondifferences may be visualized include but are not limited to,quantitative RT (reverse transcriptase), PCR, and Northern analysis.

[0138] Identification of Differentially Expressed Genes

[0139] To identify differentially expressed genes total RNA or,preferably, mRNA is isolated from tissues of interest. For example, RNAsamples are obtained from tissues of experimental subjects and fromcorresponding tissues of control subjects. Any RNA isolation techniquethat does not select against the isolation of mRNA may be utilized forthe purification of such RNA samples. See, for example, Ausubel et al.,ed., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. NewYork, 1987-1993. Large numbers of tissue samples may readily beprocessed using techniques well known to those of skill in the art, suchas, for example, the single-step RNA isolation process of Chomczynski,U.S. Pat. No. 4,843,155.

[0140] Transcripts within the collected RNA samples that represent RNAproduced by differentially expressed genes are identified by methodswell known to those of skill in the art. They include, for example,differential screening (Tedder et al., Proc. Natl. Acad. Sci. U.S.A. 85,208-12, 1988), subtractive hybridization (Hedrick et al., Nature 308,149-53; Lee et al., Proc. Natl. Acad. Sci. U.S.A. 88, 2825, 1984), anddifferential display (Liang & Pardee, Science 257, 967-71, 1992; U.S.Pat. No. 5,262,311), and microarrays.

[0141] The differential expression information may itself suggestrelevant methods for the treatment of disorders involving the humantransmembrane serine protease. For example, treatment may include amodulation of expression of the differentially expressed genes and/orthe gene encoding the human transmembrane serine protease. Thedifferential expression information may indicate whether the expressionor activity of the differentially expressed gene or gene product or thehuman transmembrane serine protease gene or gene product areup-regulated or down-regulated.

[0142] Screening Methods

[0143] The invention provides methods for identifying modulators, i.e.,candidate or test compounds which bind to transmembrane serine proteasepolypeptides or polynucleotides and/or have a stimulatory or inhibitoryeffect on, for example, expression or activity of the transmembraneserine protease polypeptide or polynucleotide, so as to regulatedegradation of the extracellular matrix. Decreased extracellular matrixdegradation is useful for preventing or suppressing malignant cells frommetastasizing. Increased extracellular matrix degradation may bedesired, for example, in developmental disorders characterized byinappropriately low levels of extracellular matrix degradation or inregeneration.

[0144] The invention provides assays for screening test compounds thatbind to or modulate the activity of a transmembrane serine proteasepolypeptide or a transmembrane serine protease polynucleotide. A testcompound preferably binds to a transmembrane serine protease polypeptideor polynucleotide. More preferably, a test compound decreases atransmembrane serine protease activity of a transmembrane serineprotease polypeptide or expression of a transmembrane serine proteasepolynucleotide by at least about 10, preferably about 50, morepreferably about 75, 90, or 100% relative to the absence of the testcompound.

[0145] Test Compounds

[0146] Test compounds can be pharmacologic agents already known in theart or can be compounds previously unknown to have any pharmacologicalactivity. The compounds can be naturally occurring or designed in thelaboratory. They can be isolated from microorganisms, animals, orplants, and can be produced recombinantly, or synthesized by chemicalmethods known in the art. If desired, test compounds can be obtainedusing any of the numerous combinatorial library methods known in theart, including but not limited to, biological libraries, spatiallyaddressable parallel solid phase or solution phase libraries, syntheticlibrary methods requiring deconvolution, the “one-bead one-compound”library method, and synthetic library methods using affinitychromatography selection. The biological library approach is limited topolypeptide libraries, while the other four approaches are applicable topolypeptide, non-peptide oligomer, or small molecule libraries ofcompounds. See Lam, Anticancer Drug Des. 12, 145, 1997.

[0147] Methods for the synthesis of molecular libraries are well knownin the art (see, for example, DeWitt et al., Proc. Natl. Acad. Sci.U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91,11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho etal., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl.33, 2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061;Gallop et al., J. Med. Chem. 37, 1233, 1994). Libraries of compounds canbe presented in solution (see, e.g., Houghten, BioTechniques 13,412-421, 1992), or on beads (Lam, Nature 354, 82-84, 1991), chips(Fodor, Nature 364, 555-556, 1993), bacteria or spores (Ladner, U.S.Pat. No. 5,223,409), plasmids (Cull et al., Proc. Natl. Acad. Sci.U.S.A. 89, 1865-1869, 1992), or phage (Scott & Smith, Science 249,386-390, 1990; Devlin, Science 249, 404-406, 1990); Cwirla et al., Proc.Natl. Acad. Sci. 97, 6378-6382, 1990; Felici, J. Mol. Biol. 222,301-310, 1991; and Ladner, U.S. Pat. No. 5,223,409).

[0148] High Throughput Screening

[0149] Test compounds can be screened for the ability to bind totransmembrane serine protease polypeptides or polynucleotides or toaffect transmembrane serine protease activity or transmembrane serineprotease gene expression using high throughput screening. Using highthroughput screening, many discrete compounds can be tested in parallelso that large numbers of test compounds can be quickly screened. Themost widely established techniques utilize 96-well microtiter plates.The wells of the microtiter plates typically require assay volumes thatrange from 50 to 500 μl. In addition to the plates, many instruments,materials, pipettors, robotics, plate washers, and plate readers arecommercially available to fit the 96-well format.

[0150] Alternatively, “free format assays,” or assays that have nophysical barrier between samples, can be used. For example, an assayusing pigment cells (melanocytes) in a simple homogeneous assay forcombinatorial peptide libraries is described by Jayawickreme et al.,Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994). The cells are placedunder agarose in petri dishes, then beads that carry combinatorialcompounds are placed on the surface of the agarose. The combinatorialcompounds are partially released the compounds from the beads. Activecompounds can be visualized as dark pigment areas because, as thecompounds diffuse locally into the gel matrix, the active compoundscause the cells to change colors.

[0151] Another example of a free format assay is described by Chelsky,“Strategies for Screening Combinatorial Libraries: Novel and TraditionalApproaches,” reported at the First Annual Conference of The Society forBiomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995). Chelskyplaced a simple homogenous enzyme assay for carbonic anhydrase inside anagarose gel such that the enzyme in the gel would cause a color changethroughout the gel. Thereafter, beads carrying combinatorial compoundsvia a photolinker were placed inside the gel and the compounds werepartially released by UV-light. Compounds that inhibited the enzyme wereobserved as local zones of inhibition having less color change.

[0152] Yet another example is described by Salmon et al., MolecularDiversity 2, 57-63 (1996). In this example, combinatorial libraries werescreened for compounds that had cytotoxic effects on cancer cellsgrowing in agar.

[0153] Another high throughput screening method is described in Beutelet al., U.S. Pat. No. 5,976,813. In this method, test samples are placedin a porous matrix. One or more assay components are then placed within,on top of, or at the bottom of a matrix such as a gel, a plastic sheet,a filter, or other form of easily manipulated solid support. Whensamples are introduced to the porous matrix they diffuse sufficientlyslowly, such that the assays can be performed without the test samplesrunning together.

[0154] Binding Assays

[0155] For binding assays, the test compound is preferably a smallmolecule that binds to and occupies the active site or a fibronectindomain of the transmembrane serine protease polypeptide, thereby makingthe active site or fibronectin domain inaccessible to substrate suchthat normal biological activity is prevented. Examples of such smallmolecules include, but are not limited to, small peptides orpeptide-like molecules. In binding assays, either the test compound orthe transmembrane serine protease polypeptide can comprise a detectablelabel, such as a fluorescent, radioisotopic, chemiluminescent, orenzymatic label, such as horseradish peroxidase, alkaline phosphatase,or luciferase. Detection of a test compound that is bound to thetransmembrane serine protease polypeptide can then be accomplished, forexample, by direct counting of radioemmission, by scintillationcounting, or by determining conversion of an appropriate substrate to adetectable product.

[0156] Alternatively, binding of a test compound to a transmembraneserine protease polypeptide can be determined without labeling either ofthe interactants. For example, a microphysiometer can be used to detectbinding of a test compound with a target polypeptide. A microphysiometer(e.g., Cytosensor™) is an analytical instrument that measures the rateat which a cell acidifies its environment using a light-addressablepotentiometric sensor (LAPS). Changes in this acidification rate can beused as an indicator of the interaction between a test compound and atransmembrane serine protease polypeptide. (McConnell et al., Science257, 1906-1912, 1992).

[0157] Determining the ability of a test compound to bind to atransmembrane serine protease polypeptide also can be accomplished usinga technology such as real-time Bimolecular Interaction Analysis (BIA).Sjolander & Urbaniczky, Anal. Chem. 63, 2338-2345, 1991, and Szabo etal., Curr. Opin. Struct. Biol. 5, 699-705, 1995. BIA is a technology forstudying biospecific interactions in real time, without labeling any ofthe interactants (e.g., BIAcore™). Changes in the optical phenomenonsurface plasmon resonance (SPR) can be used as an indication ofreal-time reactions between biological molecules.

[0158] In yet another aspect of the invention, a transmembrane serineprotease polypeptide can be used as a “bait protein” in a two-hybridassay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervoset al., Cell 72, 223-232, 1993; Madura et al., J. Biol. Chem. 268,12046-12054, 1993; Bartel et al., BioTechniques 14, 920-924, 1993;Iwabuchi et al., Oncogene 8, 1693-1696, 1993; and Brent W094/10300), toidentify other proteins which bind to or interact with the transmembraneserine protease polypeptide and modulate its activity.

[0159] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. For example, in one construct a polynucleotide encoding atransmembrane serine protease polypeptide is fused to a polynucleotideencoding the DNA binding domain of a known transcription factor (e.g.,GAL-4). In the other construct, a DNA sequence that encodes anunidentified protein (“prey” or “sample”) is fused to a polynucleotidethat codes for the activation domain of the known transcription factor.If the “bait” and the “prey” proteins are able to interact in vivo toform an protein-dependent complex, the DNA-binding and activationdomains of the transcription factor are brought into close proximity.This proximity allows transcription of a reporter gene (e.g., LacZ),which is operably linked to a transcriptional regulatory site responsiveto the transcription factor. Expression of the reporter gene can bedetected, and cell colonies containing the functional transcriptionfactor can be isolated and used to obtain the DNA sequence encoding theprotein that interacts with the transmembrane serine proteasepolypeptide.

[0160] It may be desirable to immobilize either the transmembrane serineprotease polypeptide (or polynucleotide) or the test compound tofacilitate separation of bound from unbound forms of one or both of theinteractants, as well as to accommodate automation of the assay. Thus,either the transmembrane serine protease polypeptide (or polynucleotide)or the test compound can be bound to a solid support. Suitable solidsupports include, but are not limited to, glass or plastic slides,tissue culture plates, microtiter wells, tubes, silicon chips, orparticles such as beads (including, but not limited to, latex,polystyrene, or glass beads). Any method known in the art can be used toattach the transmembrane serine protease polypeptide (or polynucleotide)or test compound to a solid support, including use of covalent andnon-covalent linkages, passive absorption, or pairs of binding moietiesattached respectively to the polypeptide or test compound and the solidsupport. Test compounds are preferably bound to the solid support in anarray, so that the location of individual test compounds can be tracked.Binding of a test compound to a transmembrane serine proteasepolypeptide (or polynucleotide) can be accomplished in any vesselsuitable for containing the reactants. Examples of such vessels includemicrotiter plates, test tubes, and microcentrifuge tubes.

[0161] In one embodiment, a transmembrane serine protease polypeptide isa fusion protein comprising a domain that allows the transmembraneserine protease polypeptide to be bound to a solid support. For example,glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe test compound or the test compound and the non-adsorbedtransmembrane serine protease polypeptide; the mixture is then incubatedunder conditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components.Binding of the interactants can be determined either directly orindirectly, as described above. Alternatively, the complexes can bedissociated from the solid support before binding is determined.

[0162] Other techniques for immobilizing polypeptides or polynucleotideson a solid support also can be used in the screening assays of theinvention. For example, either a transmembrane serine proteasepolypeptide (or polynucleotide) or a test compound can be immobilizedutilizing conjugation of biotin and streptavidin. Biotinylatedtransmembrane serine protease polypeptides or test compounds can beprepared from biotin-NHS(N-hydroxysuccinimide) using techniques wellknown in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.) and immobilized in the wells of streptavidin-coated 96 well plates(Pierce Chemical). Alternatively, antibodies which specifically bind toa transmembrane serine protease polypeptide polynucleotides, or a testcompound, but which do not interfere with a desired binding site, suchas the active site or a fibronectin domain of the transmembrane serineprotease polypeptide, can be derivatized to the wells of the plate.Unbound target or protein can be trapped in the wells by antibodyconjugation.

[0163] Methods for detecting such complexes, in addition to thosedescribed above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies which specifically bind tothe transmembrane serine protease polypeptide (or polynucleotides) ortest compound, enzyme-linked assays which rely on detecting atransmembrane serine protease activity of the transmembrane serineprotease polypeptide, and SDS gel electrophoresis under non-reducingconditions.

[0164] Screening for test compounds which bind to a transmembrane serineprotease polypeptide or polynucleotide also can be carried out in anintact cell. Any cell which comprises a transmembrane serine proteasepolynucleotide or polypeptide can be used in a cell-based assay system.A transmembrane serine protease polynucleotide can be naturallyoccurring in the cell or can be introduced using techniques such asthose described above. Either a primary culture or an established cellline, including neoplastic cell lines such as the colon cancer celllines HCT116, DLD1, HT29, Caco2, SW837, SW480, and RKO, breast cancercell lines 21-PT, 21-MT, MDA-468, SK-BR3, and BT-474, the A549 lungcancer cell line, and the H392 glioblastoma cell line, can be used. Anintact cell is contacted with a test compound. Binding of the testcompound to a transmembrane serine protease polypeptide orpolynucleotide is determined as described above, after lysing the cellto release the transmembrane serine protease polypeptide-test compoundcomplex.

[0165] Enzyme Assays

[0166] Test compounds can be tested for the ability to increase ordecrease a transmembrane serine protease activity of a transmembraneserine protease polypeptide. Transmembrane serine protease activity canbe measured, for example, using the method described in Example 2.Transmembrane serine protease activity can be measured after contactingeither a purified transmembrane serine protease polypeptide, a cellextract, or an intact cell with a test compound. A test compound thatdecreases transmembrane serine protease activity by at least about 10,preferably about 50, more preferably about 75, 90, or 100% is identifiedas a potential therapeutic agent for decreasing extracellular matrixdegradation. A test compound that increases transmembrane serineprotease activity by at least about 10, preferably about 50, morepreferably about 75, 90, or 100% is identified as a potentialtherapeutic agent for increasing extracellular matrix degradation.

[0167] Gene Expression

[0168] In another embodiment, test compounds that increase or decreasetransmembrane serine protease gene expression are identified. Atransmembrane serine protease polynucleotide is contacted with a testcompound, and the expression of an RNA or polypeptide product of thetransmembrane serine protease polynucleotide is determined. The level ofexpression of transmembrane serine protease mRNA or polypeptide in thepresence of the test compound is compared to the level of expression oftransmembrane serine protease mRNA or polypeptide in the absence of thetest compound. The test compound can then be identified as a modulatorof expression based on this comparison. For example, when expression oftransmembrane serine protease mRNA or polypeptide is greater in thepresence of the test compound than in its absence, the test compound isidentified as a stimulator or enhancer of transmembrane serine proteasemRNA or polypeptide is less expression. Alternatively, when expressionof the mRNA or protein is less in the presence of the test compound thanin its absence, the test compound is identified as an inhibitor oftransmembrane serine protease mRNA or polypeptide expression.

[0169] The level of transmembrane serine protease mRNA or polypeptideexpression in the cells can be determined by methods well known in theart for detecting mRNA or protein. Either qualitative or quantitativemethods can be used. The presence of polypeptide products of atransmembrane serine protease polynucleotide can be determined, forexample, using a variety of techniques known in the art, includingimmunochemical methods such as radioimmunoassay, Western blotting, andimmunohistochemistry. Alternatively, polypeptide synthesis can bedetermined in vivo, in a cell culture, or in an in vitro translationsystem by detecting incorporation of labeled amino acids into atransmembrane serine protease polypeptide.

[0170] Such screening can be carried out either in a cell-free assaysystem or in an intact cell. Any cell that expresses a transmembraneserine protease polynucleotide can be used in a cell-based assay system.The transmembrane serine protease polynucleotide can be naturallyoccurring in the cell or can be introduced using techniques such asthose described above. Either a primary culture or an established cellline, including neoplastic cell lines such as the colon cancer celllines HCT116, DLD1, HT29, Caco2, SW837, SW480, and RKO, breast cancercell lines 21-PT, 21-MT, MDA-468, SK-BR3, and BT-474, the A549 lungcancer cell line, and the H392 glioblastoma cell line, can be used.

[0171] Pharmaceutical Compositions

[0172] The invention also provides pharmaceutical compositions that canbe administered to a patient to achieve a therapeutic effect.Pharmaceutical compositions of the invention can comprise atransmembrane serine protease polypeptide, transmembrane serine proteasepolynucleotide, antibodies which specifically bind to a transmembraneserine protease polypeptide, or mimetics, agonists, antagonists, orinhibitors of a transmembrane serine protease polypeptide. Thecompositions can be administered alone or in combination with at leastone other agent, such as stabilizing compound, which can be administeredin any sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions can be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

[0173] In addition to the active ingredients, these pharmaceuticalcompositions can contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries that facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Pharmaceutical compositions of the invention can be administered by anynumber of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, intraperitoneal,intranasal, parenteral, topical, sublingual, or rectal means.Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

[0174] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents can be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0175] Dragee cores can be used in conjunction with suitable coatings,such as concentrated sugar solutions, which also can contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments can be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0176] Pharmaceutical preparations that can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds can be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0177] Pharmaceutical formulations suitable for parenteraladministration can be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions can contain substances that increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds can beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Non-lipid polycationic amino polymers also can be used for delivery.Optionally, the suspension also can contain suitable stabilizers oragents that increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions. For topical or nasaladministration, penetrants appropriate to the particular barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

[0178] The pharmaceutical compositions of the present invention can bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes. Thepharmaceutical composition can be provided as a salt and can be formedwith many acids, including but not limited to, hydrochloric, sulfuric,acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be moresoluble in aqueous or other protonic solvents than are the correspondingfree base forms. In other cases, the preferred preparation can be alyophilized powder which can contain any or all of the following: 1-50mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5to 5.5, that is combined with buffer prior to use.

[0179] Further details on techniques for formulation and administrationcan be found in the latest edition of REMINGTON'S PHARMACEUTICALSCIENCES (Maack Publishing Co., Easton, Pa.). After pharmaceuticalcompositions have been prepared, they can be placed in an appropriatecontainer and labeled for treatment of an indicated condition. Suchlabeling would include amount, frequency, and method of administration.

[0180] Therapeutic Indications and Methods

[0181] 1. Tumor Cell Invasion and Metastasis. Cancer is a diseasefundamentally caused by oncogenic cellular transformation. There areseveral hallmarks of transformed cells that distinguish them from theirnormal counterparts and underlie the pathophysiology of cancer. Theseinclude uncontrolled cellular proliferation, unresponsiveness to normaldeath-inducing signals (immortalization), increased cellular motilityand invasiveness, increased ability to recruit blood supply throughinduction of new blood vessel formation (angiogenesis), geneticinstability, and dysregulated gene expression. Various combinations ofthese aberrant physiologies, along with the acquisition ofdrug-resistance frequently lead to an intractable disease state in whichorgan failure and patient death ultimately ensue.

[0182] Most standard cancer therapies target cellular proliferation andrely on the differential proliferative capacities between transformedand normal cells for their efficacy. This approach is hindered by thefacts that several important normal cell types are also highlyproliferative and that cancer cells frequently become resistant to theseagents. Thus, the therapeutic indices for traditional anti-cancertherapies rarely exceed 2.0.

[0183] The advent of genomics-driven molecular target identification hasopened up the possibility of identifying new cancer-specific targets fortherapeutic intervention that will provide safer, more effectivetreatments for cancer patients. Thus, newly discovered tumor-associatedgenes and their products can be tested for their role(s) in disease andused as tools to discover and develop innovative therapies. Genesplaying important roles in any of the physiological processes outlinedabove can be characterized as cancer targets.

[0184] Genes or gene fragments identified through genomics can readilybe expressed in one or more heterologous expression systems to producefunctional recombinant proteins. These proteins are characterized invitro for their biochemical properties and then used as tools inhigh-throughput molecular screening programs to identify chemicalmodulators of their biochemical activities. Agonists and/or antagonistsof target protein activity can be identified in this manner andsubsequently tested in cellular and in vivo disease models foranti-cancer activity. Optimization of lead compounds with iterativetesting in biological models and detailed pharmacokinetic andtoxicological analyses form the basis for drug development andsubsequent testing in humans.

[0185] The human transmembrane serine protease gene provides atherapeutic target for decreasing extracellular matrix degradation, inparticular for treating or preventing metastatic cancer. For example,blocking a fibronectin domain of human ephrin-like serine protease cansuppress or prevent migration or metastasis of tumor cells in responseto fibronectin (9, 10). Cancers whose metastasis can be suppressedaccording to the invention include adenocarcinoma, melanoma, cancers ofthe adrenal gland, bladder, bone, breast, cervix, gall bladder, liver,lung, ovary, pancreas, prostate, testis, and uterus. Circulating tumorcells arrested in the capillary beds of different organs must invade theendothelial cell lining and degrade its underlying basement membrane(BM) in order to invade into the extravascular tissue(s) where theyestablish metastasis (1, 2). Metastatic tumor cells often attach at ornear the intercellular junctions between adjacent endothelial cells.Such attachment of the metastatic cells is followed by rupture of thejunctions, retraction of the endothelial cell borders and migrationthrough the breach in the endothelium toward the exposed underlying BM(1, 11).

[0186] Once located between endothelial cells and the BM, the invadingcells must degrade the subendothelial glycoproteins and proteoglycans ofthe BM in order to migrate out of the vascular compartment. Severalcellular enzymes (e.g., collagenase IV, plasminogen activator, cathepsinB, elastase) are thought to be involved in degradation of BM (2, 11).Suppression of human transmembrane serine protease activity thereforecan be used to suppress tumor cell invasion and metastasis.

[0187] 2. Tumor Angiogenesis. Basic fibroblast growth factor (bFGF) hasbeen extracted from the subendothelial extracellular matrix produced invitro (3) and from basement membranes of the cornea (4), suggesting thatextracellular matrix may serve as a reservoir for bFGF.Immunohistochemical staining revealed the localization of bFGF inbasement membranes of diverse tissues and blood vessels (5). Despite theubiquitous presence of bFGF in normal tissues, endothelial cellproliferation in these tissues is usually very low, which suggests thatbFGF is somehow sequestered from its site of action. It is possible,therefore, that suppression of human transmembrane serine proteaseactivity can suppress release of active bFGF from extracellular matrixand basement membranes. In addition, displacement of bFGF from itsstorage within basement membranes and extracellular matrix may thereforeprovide a novel mechanism for induction of neovascularization in normaland pathological situations. Restriction of endothelial cell growthfactors in the extracellular matrix may prevent their systemic action onthe vascular endothelium, thus maintaining a very low rate ofendothelial cells turnover and vessel growth. On the other hand, releaseof bFGF from storage in the extracellular matrix may elicit localizedendothelial cell proliferation and neovascularization in processes suchas wound healing, inflammation and tumor development (6, 7).

[0188] 3. Inflammation and Cellular Immunity. Transmembrane serineprotease activity may be involved in the ability of activated cells ofthe immune system to leave the circulation and elicit both inflammatoryand autoimmune responses. Thus, inflammation and cellular immunity maybe regulated by regulating activity of transmembrane serine protease.

[0189] 4. Viral infection. Removal of the cell surface components bytransmembrane serine protease may influence the ability of viruses toattach to the cell surface. Regulation of transmembrane serine proteasemay therefore be used to treat viral infections.

[0190] 5. Neurodegenerative diseases. It is also possible thattransmembrane serine protease activity can be used to degrade, forexample, prion protein amyloid plaques of Genstmann-Straussler Syndrome,Creutzfeldt-Jakob disease, and Scrapie.

[0191] CNS disorders which may be treated include brain injuries,cerebrovascular diseases and their consequences, Parkinson's disease,corticobasal degeneration, motor neuron disease, dementia, includingALS, multiple sclerosis, traumatic brain injury, stroke, post-stroke,post-traumatic brain injury, and small-vessel cerebrovascular disease.Dementias, such as Alzheimer's disease, vascular dementia, dementia withLewy bodies, frontotemporal dementia and Parkinsonism linked tochromosome 17, frontotemporal dementias, including Pick's disease,progressive nuclear palsy, corticobasal degeneration, Huntington'sdisease, thalamic degeneration, Creutzfeld-Jakob dementia, HIV dementia,schizophrenia with dementia, and Korsakoff's psychosis also can betreated. Similarly, it may be possible to treat cognitive-relateddisorders, such as mild cognitive impairment, age-associated memoryimpairment, age-related cognitive decline, vascular cognitiveimpairment, attention deficit disorders, attention deficit hyperactivitydisorders, and memory disturbances in children with learningdisabilities, by regulating the activity of human transmembrane serineprotease.

[0192] Pain that is associated with CNS disorders also can be treated byregulating the activity of human transmembrane serine protease. Painwhich can be treated includes that associated with central nervoussystem disorders, such as multiple sclerosis, spinal cord injury,sciatica, failed back surgery syndrome, traumatic brain injury,epilepsy, Parkinson's disease, post-stroke, and vascular lesions in thebrain and spinal cord (e.g., infarct, hemorrhage, vascularmalformation). Non-central neuropathic pain includes that associatedwith post mastectomy pain, reflex sympathetic dystrophy (RSD),trigeminal neuralgiaradioculopathy, post-surgical pain, HIV/AIDS relatedpain, cancer pain, metabolic neuropathies (e.g., diabetic neuropathy,vasculitic neuropathy secondary to connective tissue disease),paraneoplastic polyneuropathy associated, for example, with carcinoma oflung, or leukemia, or lymphoma, or carcinoma of prostate, colon orstomach, trigeminal neuralgia, cranial neuralgias, and post-herpeticneuralgia. Pain associated with cancer and cancer treatment also can betreated, as can headache pain (for example, migraine with aura, migrainewithout aura, and other migraine disorders), episodic and chronictension-type headache, tension-type like headache, cluster headache, andchronic paroxysmal hemicrania.

[0193] 6. Restenosis and Atherosclerosis. Proliferation of arterialsmooth muscle cells (SMCs) in response to endothelial injury andaccumulation of cholesterol rich lipoproteins are basic events in thepathogenesis of atherosclerosis and restenosis (8). It is possible thattransmembrane serine protease may be involved in the catabolic pathwaythat may allow substantial cellular and interstitial accumulation ofcholesterol rich lipoproteins. The latter pathway is expected to behighly atherogenic by promoting accumulation of apoB and apoE richlipoproteins (i.e. LDL, VLDL, chylomicrons), independent of feedbackinhibition by the cellular sterol content. Altered levels of humantransmembrane serine protease activity therefore may inhibit both SMCproliferation and lipid accumulation and thus may halt the progressionof restenosis and atherosclerosis.

[0194] 7. COPD. Chronic obstructive pulmonary (or airways) disease(COPD) is a condition defined physiologically as airflow obstructionthat generally results from a mixture of emphysema and peripheral airwayobstruction due to chronic bronchitis (Senior & Shapiro, PulmonaryDiseases and Disorders, 3d ed., New York, McGraw-Hill, 1998, pp.659-681, 1998; Barnes, Chest 117, 10S-14S, 2000). Emphysema ischaracterized by destruction of alveolar walls leading to abnormalenlargement of the air spaces of the lung. Chronic bronchitis is definedclinically as the presence of chronic productive cough for three monthsin each of two successive years. In COPD, airflow obstruction is usuallyprogressive and is only partially reversible. By far the most importantrisk factor for development of COPD is cigarette smoking, although thedisease does occur in non-smokers.

[0195] Chronic inflammation of the airways is a key pathological featureof COPD (Senior & Shapiro, 1998). The inflammatory cell populationcomprises increased numbers of macrophages, neutrophils, and CD8⁺lymphocytes. Inhaled irritants, such as cigarette smoke, activatemacrophages that are resident in the respiratory tract, as well asepithelial cells leading to release of chemokines (e.g., interleukin-8)and other chemotactic factors. These chemotactic factors act to increasethe neutrophil/monocyte trafficking from the blood into the lung tissueand airways. Neutrophils and monocytes recruited into the airways canrelease a variety of potentially damaging mediators such as proteolyticenzymes and reactive oxygen species. Matrix degradation and emphysema,along with airway wall thickening, surfactant dysfunction, and mucushypersecretion, all are potential sequelae of this inflammatory responsethat lead to impaired airflow and gas exchange.

[0196] COPD is characterized by damage to the lung extracellular matrixand emphysema can be viewed as the pathologic process that affects thelung parenchyma. This process eventually leads to the destruction of theairway walls resulting in permanent airspace enlargement (Senior andShapiro, in PULMONARY DISEASES AND DISORDERS, 3^(rd) ed., New York,McGraw-Hill, 1998, pp. 659-681, 1998). The observation that inheriteddeficiency of a1-antitrypsin (a1-AT), the primary inhibitor ofneutrophil elastase, predisposes individuals to early onset emphysema,and that intrapulmonary instillation of elastolytic enzymes inexperimental animals causes emphysema, led to the elastase:antielastasehypothesis for the pathogenesis of emphysema (Eriksson, Acta Med. Scand.177(Suppl.), 432, 1965, Gross, J. Occup. Med. 6, 481-84, 1964). This inturn led to the concept that destruction of elastin in the lungparenchyma is the basis of the development of emphysema.

[0197] A broad range of immune and inflammatory cells includingneutrophils, macrophages, T lymphocytes and eosinophils containproteolytic enzymes that could contribute to the destruction of lungextracellular matrix (Shapiro, 1999). In addition, a number of differentclasses of proteases have been identified that have the potential tocontribute to lung matrix destruction. These include serine proteases,matrix metalloproteinases and cysteine proteases. Of these classes ofenzymes, a number can hydrolyze elastin and have been shown to beelevated in COPD patients (neutrophil elastase, MMP-2, 9, 12) (Culpittet al., Am. J. Respir. Crit. Care Med. 160, 1635-39, 1999, Shapiro, Am.J. Crit. Care Med. 160 (5), S29-S32, 1999).

[0198] It is expected that in the future novel members of the existingclasses of proteases and new classes of proteases will be identifiedthat play a significant role in the damage of the extracellular lungmatrix including elastin proteolysis. Novel protease targets thereforeremain very attractive therapeutic targets.

[0199] 8. Other therapeutic and diagnostic indications. Anti-humantransmembrane serine protease antibodies can be applied forimmunodetection and diagnosis of micrometastases, autoimmune lesions,and renal failure in biopsy specimens, plasma samples, and body fluids.Alternatively, if desired a transmembrane serine protease function canbe supplied to a cell by introducing a transmembrane serineprotease-encoding polynucleotide into the cell.

[0200] The invention further pertains to the use of novel agentsidentified by the screening assays described above. Accordingly, it iswithin the scope of this invention to use a test compound identified asdescribed herein in an appropriate animal model. For example, an agentidentified as described herein (e.g., a modulating agent, an antisensenucleic acid molecule, a specific antibody, ribozyme, or apolypeptide-binding partner) can be used in an animal model to determinethe efficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal model to determine the mechanism of action of such an agent.Furthermore, this invention pertains to uses of novel agents identifiedby the above-described screening assays for treatments as describedherein.

[0201] A reagent which affects transmembrane serine protease activitycan be administered to a human cell, either in vitro or in vivo, toreduce transmembrane serine protease activity. The reagent preferablybinds to an expression product of a human transmembrane serine proteasegene. If the expression product is a polypeptide, the reagent ispreferably an antibody. For treatment of human cells ex vivo, anantibody can be added to a preparation of stem cells that have beenremoved from the body. The cells can then be replaced in the same oranother human body, with or without clonal propagation, as is known inthe art.

[0202] In one embodiment, the reagent is delivered using a liposome.Preferably, the liposome is stable in the animal into which it has beenadministered for at least about 30 minutes, more preferably for at leastabout 1 hour, and even more preferably for at least about 24 hours. Aliposome comprises a lipid composition that is capable of targeting areagent, particularly a polynucleotide, to a particular site in ananimal, such as a human. Preferably, the lipid composition of theliposome is capable of targeting to a specific organ of an animal, suchas the lung or liver.

[0203] A liposome useful in the present invention comprises a lipidcomposition that is capable of fusing with the plasma membrane of thetargeted cell to deliver its contents to the cell. Preferably, thetransfection efficiency of a liposome is about 0.5 μg of DNA per 16nmole of liposome delivered to about 10⁶ cells, more preferably about1.0 μg of DNA per 16 nmol of liposome delivered to about 10⁶ cells, andeven more preferably about 2.0 μg of DNA per 16 nmol of liposomedelivered to about 10⁶ cells. Preferably, a liposome is between about100 and 500 nm, more preferably between about 150 and 450 nm, and evenmore preferably between about 200 and 400 nm in diameter.

[0204] Suitable liposomes for use in the present invention include thoseliposomes standardly used in, for example, gene delivery methods knownto those of skill in the art. More preferred liposomes include liposomeshaving a polycationic lipid composition and/or liposomes having acholesterol backbone conjugated to polyethylene glycol. Optionally, aliposome comprises a compound capable of targeting the liposome to atumor cell, such as a tumor cell ligand exposed on the outer surface ofthe liposome.

[0205] Complexing a liposome with a reagent such as an antisenseoligonucleotide or ribozyme can be achieved using methods that arestandard in the art (see, for example, U.S. Pat. No. 5,705,151).Preferably, from about 0.1 μg to about 10 μg of polynucleotide iscombined with about 8 nmol of liposomes, more preferably from about 0.5μg to about 5 μg of polynucleotides are combined with about 8 nmolliposomes, and even more preferably about 1.0 μg of polynucleotides iscombined with about 8 nmol liposomes.

[0206] In another embodiment, antibodies can be delivered to specifictissues in vivo using receptor-mediated targeted delivery.Receptor-mediated DNA delivery techniques are taught in, for example,Findeis et al. Trends in Biotechnol. 11, 202-05 (1993); Chiou et al.,GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu etal., J. Biol. Chem. 269, 542-46 (1994); Zenke et al., Proc. Natl. Acad.Sci. U.S.A. 87, 3655-59 (1990); Wu et al., J. Biol. Chem. 266, 338-42(1991).

[0207] If the reagent is a single-chain antibody, polynucleotidesencoding the antibody can be constructed and introduced into a celleither ex vivo or in vivo using well-established techniques including,but not limited to, transferrin-polycation-mediated DNA transfer,transfection with naked or encapsulated nucleic acids, liposome-mediatedcellular fusion, intracellular transportation of DNA-coated latex beads,protoplast fusion, viral infection, electroporation, “gene gun,” andDEAE- or calcium phosphate-mediated transfection.

[0208] Determination of a Therapeutically Effective Dose

[0209] The determination of a therapeutically effective dose is wellwithin the capability of those skilled in the art. A therapeuticallyeffective dose refers to that amount of active ingredient that increasesor decreases extracellular matrix degradation relative to that whichoccurs in the absence of the therapeutically effective dose.

[0210] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays or in animal models,usually mice, rabbits, dogs, or pigs. The animal model also can be usedto determine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans.

[0211] Therapeutic efficacy and toxicity, e.g., ED₅₀ (the dosetherapeutically effective in 50% of the population) and LD₅₀ (the doselethal to 50% of the population), can be determined by standardpharmaceutical procedures in cell cultures or experimental animals. Thedose ratio of toxic to therapeutic effects is the therapeutic index, andit can be expressed as the ratio, LD₅₀/ED₅₀.

[0212] Pharmaceutical compositions that exhibit large therapeuticindices are preferred. The data obtained from cell culture assays andanimal studies is used in formulating a range of dosage for human use.The dosage contained in such compositions is preferably within a rangeof circulating concentrations that include the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0213] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activeingredient or to maintain the desired effect. Factors that can be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions can be administered every 3 to 4 days, everyweek, or once every two weeks depending on the half-life and clearancerate of the particular formulation.

[0214] Normal dosage amounts can vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0215] Effective in vivo dosages of an antibody are in the range ofabout 5 μg to about 50 μg/kg, about 50 μg to about 5 mg/kg, about 100 μgto about 500 μg/kg of patient body weight, and about 200 to about 250μg/kg of patient body weight. For administration of polynucleotidesencoding single-chain antibodies, effective in vivo dosages are in therange of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μgto about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100μg of DNA.

[0216] If the expression product is mRNA, the reagent is preferably anantisense oligonucleotide or a ribozyme. Polynucleotides that expressantisense oligonucleotides or ribozymes can be introduced into cells bya variety of methods, as described above.

[0217] Preferably, a reagent reduces expression of a transmembraneserine protease polynucleotide or activity of a transmembrane serineprotease polypeptide by at least about 10, preferably about 50, morepreferably about 75, 90, or 100% relative to the absence of the reagent.The effectiveness of the mechanism chosen to decrease the level ofexpression of a transmembrane serine protease polynucleotide or theactivity of a transmembrane serine protease polypeptide can be assessedusing methods well known in the art, such as hybridization of nucleotideprobes to transmembrane serine protease-specific mRNA, quantitativeRT-PCR, immunologic detection of a transmembrane serine proteasepolypeptide, or measurement of transmembrane serine protease activity.

[0218] In any of the embodiments described above, any of thepharmaceutical compositions of the invention can be administered incombination with other appropriate therapeutic agents. Selection of theappropriate agents for use in combination therapy can be made by one ofordinary skill in the art, according to conventional pharmaceuticalprinciples. The combination of therapeutic agents can actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

[0219] Any of the therapeutic methods described above can be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0220] The above disclosure generally describes the present invention,and all patents and patent applications cited in this disclosure areexpressly incorporated herein. A more complete understanding can beobtained by reference to the following specific examples, which areprovided for purposes of illustration only and are not intended to limitthe scope of the invention.

EXAMPLE 1 Identification of a Test Compound that Binds to aTransmembrane Serine Protease Polypeptide

[0221] Purified transmembrane serine protease polypeptides comprising aglutathione-S-transferase protein and absorbed ontoglutathione-derivatized wells of 96-well microtiter plates are contactedwith test compounds from a small molecule library at pH 7.0 in aphysiological buffer solution. Transmembrane serine proteasepolypeptides comprise an amino acid sequence shown in SEQ ID NO:12 Thetest compounds comprise a fluorescent tag. The samples are incubated for5 minutes to one hour. Control samples are incubated in the absence of atest compound.

[0222] The buffer solution containing the test compounds is washed fromthe wells. Binding of a test compound to a transmembrane serine proteasepolypeptide is detected by fluorescence measurements of the contents ofthe wells. A test compound that increases the fluorescence in a well byat least 15% relative to fluorescence of a well in which a test compoundwas not incubated is identified as a compound that binds to atransmembrane serine protease polypeptide.

EXAMPLE 2 Identification of a Test Compound which DecreasesTransmembrane Serine Protease Activity

[0223] Cellular extracts from the human colon cancer cell line HCT116are contacted with test compounds from a small molecule library andassayed for transmembrane serine protease activity. Control extracts, inthe absence of a test compound, also are assayed. Protease activity canbe measured using thiobenzylester substrates, as described in U.S. Pat.No. 5,500,344. For monitoring enzyme activities from granules and columnfractions, assays are performed at room temperature using 0.5 mM5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) (Sigma) to detect the HSBzlleaving group (ε₄₁₀=13600 M⁻¹ cm⁻¹).

[0224] BLT-esterase activity is estimated using a microtiter assay(Green and Shaw, Anal. Biochem. 93, 223-226, 1979). Briefly, 50 μl ofsample is added to 100 μl of 1 mM DTNB, made up in 10 mM HEPES, 1 mMCaCl₂, 1 mM MgCl₂, pH 7.2. The reaction is initiated by the addition of50 μl of BLT (Sigma) to give a final concentration of 500 μM. For Metasedeterminations, 50 μl of dilutions of the sample in 0.1 M HEPES, 0.05 MCaCl₂, pH 7.5, are added to 100 μl of 1 mM DTNB, and the reaction isinitiated by the addition of 50 μl of Boc-Ala-Ala-Met-S Benzyl (Bzl) togive a final concentration of 150 μM. The duration of the assay dependson color development, the rate of which is measured (O.D.₄₁₀) on aDynatech MR 5000 microplate reader. Controls of sample and DTNB alone orDTNB and substrate alone are run.

[0225] For more sensitive comparisons of enzymatic activities, peptidethiobenzyl ester substrates are used to measure protease activities. Thechymase substrate Suc-Phe-Leu-Phe-SBzl is purchased from BACHEMBioscience Inc., Philadelphia, Pa. Z-Arg-SBzl (the tryptase substrate,Kam et al., J. Biol. Chem. 262, 3444-3451, 1987); Boc-Ala-Ala-AA-SBzl(AA=Asp, Met, Leu, Nle, or Ser), and Suc-Ala-Ala-Met-SBzl (Odake et al,Biochemistry 30, 2217-2227, 1991); Harper et al., Biochemistry 23,2995-3002, 1984) are synthesized previously. Boc-Ala-Ala-Asp-SBzl is thesubstrate for Asp-ase and peptide thiobenzyl esters containing Met, Leuor Nle are substrates for Met-ase SP. Assays are performed at roomtemperature in 0.1 M, HEPES buffer, pH 7.5, containing 0.01 M CaCl₂ and8% Me₂O using 0.34 mM 4,4′-dithiodipyridine (Aldrithiol-4, AldrichChemical Co., Milwaukee, Wis.) to detect HSBzl leaving group that reactswith 4,4′-dithiodipyridine to release thiopyridone (ε324=19800 M⁻¹ cm⁻¹,Grasetti and Murray, Arch. Biochem. Biophys. 119, 41-49, 1967). Theinitial rates are measured at 324 nm using a Beckman 35spectrophotometer when 10-25 μl of an enzyme stock solution is added toa cuvette containing 2.0 ml of buffer, 150 μl of 4,4′-dithiodipyridine,and 25 μl of substrate. The same volume of substrate and4,4′-dithiodipyridine are added to the reference cell in order tocompensate for the background hydrolysis rate of the substrates. Initialrates are measured in duplicate for each substrate concentration and areaveraged in each case. Substrate concentrations are 100-133 μM.

[0226] A test compound that decreases transmembrane serine proteaseactivity of the extract relative to the control extract by at least 20%is identified as a transmembrane serine protease inhibitor.

EXAMPLE 3 Identification of a Test Compound which DecreasesTransmembrane Serine Protease Gene Expression

[0227] A test compound is administered to a culture of the breast tumorcell line MDA-468 and incubated at 37° C. for 10 to 45 minutes. Aculture of the same type of cells incubated for the same time withoutthe test compound provides a negative control.

[0228] RNA is isolated from the two cultures as described in Chirgwin etal., Biochem. 18, 5294-99, 1979). Northern blots are prepared using 20to 30 μg total RNA and hybridized with a ³²P-labeled transmembraneserine protease-specific probe at 65° C. in Express-hyb (CLONTECH). Theprobe comprises at least 11 contiguous nucleotides selected from thecomplement of SEQ ID NO:11. A test compound that decreases thetransmembrane serine protease -specific signal relative to the signalobtained in the absence of the test compound is identified as aninhibitor of transmembrane serine protease gene expression.

EXAMPLE 4 Treatment of a Breast Tumor with a Reagent that SpecificallyBinds to a Transmembrane Serine Protease Gene Product

[0229] Synthesis of antisense transmembrane serine proteaseoligonucleotides comprising at least 11 contiguous nucleotides selectedfrom the complement of SEQ ID NO:11 is performed on a Pharmacia GeneAssembler series synthesizer using the phosphoramidite procedure(Uhlmann et al., Chem. Rev. 90, 534-83, 1990). Following assembly anddeprotection, oligonucleotides are ethanol-precipitated twice, dried,and suspended in phosphate-buffered saline (PBS) at the desiredconcentration. Purity of these oligonucleotides is tested by capillarygel electrophoreses and ion exchange HPLC. Endotoxin levels in theoligonucleotide preparation are determined using the Limulus AmebocyteAssay (Bang, Biol. Bull. (Woods Hole, Mass.) 105, 361-362, 1953).

[0230] An aqueous composition containing the antisense oligonucleotidesat a concentration of 0.1-100 μM is injected directly into a breasttumor with a needle. The needle is placed in the tumors and withdrawnwhile expressing the aqueous composition within the tumor.

[0231] The breast tumor is monitored over a period of days or weeks.Additional injections of the antisense oligonucleotides can be givenduring that time. Metastasis of the breast tumor is suppressed due todecreased transmembrane serine protease activity of the breast tumorcells.

EXAMPLE 5 Expression of Recombinant Human Transmembrane Serine Protease

[0232] The Pichia pastoris expression vector pPICZB (Invitrogen, SanDiego, Calif.) is used to produce large quantities of recombinant humantransmembrane serine protease polypeptides in yeast. The transmembraneserine protease -encoding DNA sequence is derived from SEQ ID NO:11.Before insertion into vector pPICZB, the DNA sequence is modified bywell-known methods in such a way that it contains at its 5′-end aninitiation codon and at its 3′-end an enterokinase cleavage site, a His6reporter tag and a termination codon. Moreover, at both terminirecognition sequences for restriction endonucleases are added and afterdigestion of the multiple cloning site of pPICZ B with the correspondingrestriction enzymes the modified DNA sequence is ligated into pPICZB.This expression vector is designed for inducible expression in Pichiapastoris, driven by a yeast promoter. The resulting pPICZ/md-His6 vectoris used to transform the yeast.

[0233] The yeast is cultivated under usual conditions in 5 liter shakeflasks and the recombinantly produced protein isolated from the cultureby affinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea.The bound polypeptide is eluted with buffer, pH 3.5, and neutralized.Separation of the polypeptide from the His6 reporter tag is accomplishedby site-specific proteolysis using enterokinase (Invitrogen, San Diego,Calif.) according to manufacturer's instructions. Purified humantransmembrane serine protease polypeptide is obtained.

EXAMPLE 6 Proliferation Inhibition Assay: Antisense OligonucleotidesSuppress the Growth of Cancer Cell Lines

[0234] The cell line used for testing is the human colon cancer cellline HCT116. Cells are cultured in RPMI-1640 with 10-15% fetal calfserum at a concentration of 10,000 cells per milliliter in a volume of0.5 ml and kept at 37° C. in a 95% air/5% CO₂ atmosphere.

[0235] Phosphorothioate oligoribonucleotides are synthesized on anApplied Biosystems Model 380B DNA synthesizer using phosphoroamiditechemistry. A sequence of 24 bases complementary to the nucleotides atposition 1 to 24 of SEQ ID NO:11 is used as the test oligonucleotide. Asa control, another (random) sequence is used: 5′-TCA ACT GAC TAG ATG TACATG GAC-3′ (SEQ ID NO:36). Following assembly and deprotection,oligonucleotides are ethanol-precipitated twice, dried, and suspended inphosphate buffered saline at the desired concentration. Purity of theoligonucleotides is tested by capillary gel electrophoresis and ionexchange HPLC. The purified oligonucleotides are added to the culturemedium at a concentration of 10 μM once per day for seven days.

[0236] The addition of the test oligonucleotide for seven days resultsin significantly reduced expression of human transmembrane serineprotease as determined by Western blotting. This effect is not observedwith the control oligonucleotide. After 3 to 7 days, the number of cellsin the cultures is counted using an automatic cell counter. The numberof cells in cultures treated with the test oligonucleotide (expressed as100%) is compared with the number of cells in cultures treated with thecontrol oligonucleotide. The number of cells in cultures treated withthe test oligonucleotide is not more than 30% of control, indicatingthat the inhibition of human transmembrane serine protease has ananti-proliferative effect on cancer cells.

EXAMPLE 7 In Vivo Testing of Compounds/Target Validation

[0237] 1. Acute Mechanistic Assays

[0238] 1.1 Reduction in Mitogenic Plasma Hormone Levels

[0239] This non-tumor assay measures the ability of a compound to reduceeither the endogenous level of a circulating hormone or the level ofhormone produced in response to a biologic stimulus. Rodents areadministered test compound (p.o., i.p., i.v., i.m., or s.c.). At apredetermined time after administration of test compound, blood plasmais collected. Plasma is assayed for levels of the hormone of interest.If the normal circulating levels of the hormone are too low and/orvariable to provide consistent results, the level of the hormone may beelevated by a pre-treatment with a biologic stimulus (i.e., LHRH may beinjected i.m. into mice at a dosage of 30 ng/mouse to induce a burst oftestosterone synthesis). The timing of plasma collection would beadjusted to coincide with the peak of the induced hormone response.Compound effects are compared to a vehicle-treated control group. AnF-test is preformed to determine if the variance is equal or unequalfollowed by a Student's t-test. Significance is p value≦0.05 compared tothe vehicle control group.

[0240] 1.2. Hollow Fiber Mechanism of Action Assay

[0241] Hollow fibers are prepared with desired cell line(s) andimplanted intraperitoneally and/or subcutaneously in rodents. Compoundsare administered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested inaccordance with specific readout assay protocol, these may includeassays for gene expression (bDNA, PCR, or Taqman), or a specificbiochemical activity (i.e., cAMP levels. Results are analyzed byStudent's t-test or Rank Sum test after the variance between groups iscompared by an F-test, with significance at p≦0.05 as compared to thevehicle control group.

[0242] 2. Subacute Functional In Vivo Assays

[0243] 2.1. Reduction in Mass of Hormone Dependent Tissues

[0244] This is another non-tumor assay that measures the ability of acompound to reduce the mass of a hormone dependent tissue (i.e., seminalvesicles in males and uteri in females). Rodents are administered testcompound (p.o., i.p., i.v., i.m., or s.c.) according to a predeterminedschedule and for a predetermined duration (i.e., 1 week). At terminationof the study, animals are weighed, the target organ is excised, anyfluid is expressed, and the weight of the organ is recorded. Bloodplasma may also be collected. Plasma may be assayed for levels of ahormone of interest or for levels of test agent. Organ weights may bedirectly compared or they may be normalized for the body weight of theanimal. Compound effects are compared to a vehicle-treated controlgroup. An F-test is preformed to determine if the variance is equal orunequal followed by a Student's t-test. Significance is p value≦0.05compared to the vehicle control group.

[0245] 2.2. Hollow Fiber Proliferation Assay

[0246] Hollow fibers are prepared with desired cell line(s) andimplanted intraperitoneally and/or subcutaneously in rodents. Compoundsare administered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested inaccordance with specific readout assay protocol. Cell proliferation isdetermined by measuring a marker of cell number (i.e., MTT or LDH). Thecell number and change in cell number from the starting inoculum areanalyzed by Student's t-test or Rank Sum test after the variance betweengroups is compared by an F-test, with significance at p≦0.05 as comparedto the vehicle control group.

[0247] 2.3. Anti-angiogenesis Models

[0248] 2.3.1. Corneal Angiogenesis

[0249] Hydron pellets with or without growth factors or cells areimplanted into a micropocket surgically created in the rodent cornea.Compound administration may be systemic or local (compound mixed withgrowth factors in the hydron pellet). Corneas are harvested at 7 dayspost implantation immediately following intracardiac infusion ofcolloidal carbon and are fixed in 10% formalin. Readout is qualitativescoring and/or image analysis. Qualitative scores are compared by RankSum test. Image analysis data is evaluated by measuring the area ofneovascularization (in pixels) and group averages are compared byStudent's t-test (2 tail). Significance is p≦0.05 as compared to thegrowth factor or cells only group.

[0250] 2.3.2. Matrigel Angiogenesis

[0251] Matrigel, containing cells or growth factors, is injectedsubcutaneously. Compounds are administered p.o., i.p., i.v., i.m., ors.c. Matrigel plugs are harvested at predetermined time point(s) andprepared for readout. Readout is an ELISA-based assay for hemoglobinconcentration and/or histological examination (i.e. vessel count,special staining for endothelial surface markers: CD31, factor-8).Readouts are analyzed by Student's t-test, after the variance betweengroups is compared by an F-test, with significance determined at p≦0.05as compared to the vehicle control group.

[0252] 3. Primary Antitumor Efficacy

[0253] 3.1. Early Therapy Models

[0254] 3.1.1. Subcutaneous Tumor

[0255] Tumor cells or fragments are implanted subcutaneously on Day 0.Vehicle and/or compounds are administered p.o., i.p., i.v., i.m., ors.c. according to a predetermined schedule starting at a time, usuallyon Day 1, prior to the ability to measure the tumor burden. Body weightsand tumor measurements are recorded 2-3 times weekly. Mean net body andtumor weights are calculated for each data collection day. Anti-tumorefficacy may be initially determined by comparing the size of treated(T) and control (C) tumors on a given day by a Student's t-test, afterthe variance between groups is compared by an F-test, with significancedetermined at p≦0.05. The experiment may also be continued past the endof dosing in which case tumor measurements would continue to be recordedto monitor tumor growth delay. Tumor growth delays are expressed as thedifference in the median time for the treated and control groups toattain a predetermined size divided by the median time for the controlgroup to attain that size. Growth delays are compared by generatingKaplan-Meier curves from the times for individual tumors to attain theevaluation size. Significance is p≦0.05.

[0256] 3.1.2. Intraperitoneal/Intracranial Tumor Models

[0257] Tumor cells are injected intraperitoneally or intracranially onDay 0. Compounds are administered p.o., i.p., i.v., i.m., or s.c.according to a predetermined schedule starting on Day 1. Observations ofmorbidity and/or mortality are recorded twice daily. Body weights aremeasured and recorded twice weekly. Morbidity/mortality data isexpressed in terms of the median time of survival and the number oflong-term survivors is indicated separately. Survival times are used togenerate Kaplan-Meier curves. Significance is p≦0.05 by a log-rank testcompared to the control group in the experiment.

[0258] 3.2. Established Disease Model

[0259] Tumor cells or fragments are implanted subcutaneously and grownto the desired size for treatment to begin. Once at the predeterminedsize range, mice are randomized into treatment groups. Compounds areadministered p.o., i.p., i.v., i.m., or s.c. according to apredetermined schedule. Tumor and body weights are measured and recorded2-3 times weekly. Mean tumor weights of all groups over days postinoculation are graphed for comparison. An F-test is preformed todetermine if the variance is equal or unequal followed by a Student'st-test to compare tumor sizes in the treated and control groups at theend of treatment. Significance is p≦0.05 as compared to the controlgroup. Tumor measurements may be recorded after dosing has stopped tomonitor tumor growth delay. Tumor growth delays are expressed as thedifference in the median time for the treated and control groups toattain a predetermined size divided by the median time for the controlgroup to attain that size. Growth delays are compared by generatingKaplan-Meier curves from the times for individual tumors to attain theevaluation size. Significance is p value≦0.05 compared to the vehiclecontrol group.

[0260] 3.3. Orthotopic Disease Models

[0261] 3.3.1. Mammary Fat Pad Assay

[0262] Tumor cells or fragments, of mammary adenocarcinoma origin, areimplanted directly into a surgically exposed and reflected mammary fatpad in rodents. The fat pad is placed back in its original position andthe surgical site is closed. Hormones may also be administered to therodents to support the growth of the tumors. Compounds are administeredp.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule.Tumor and body weights are measured and recorded 2-3 times weekly. Meantumor weights of all groups over days post inoculation are graphed forcomparison. An F-test is preformed to determine if the variance is equalor unequal followed by a Student's t-test to compare tumor sizes in thetreated and control groups at the end of treatment. Significance isp≦0.05 as compared to the control group.

[0263] Tumor measurements may be recorded after dosing has stopped tomonitor tumor growth delay. Tumor growth delays are expressed as thedifference in the median time for the treated and control groups toattain a predetermined size divided by the median time for the controlgroup to attain that size. Growth delays are compared by generatingKaplan-Meier curves from the times for individual tumors to attain theevaluation size. Significance is p value≦0.05 compared to the vehiclecontrol group. In addition, this model provides an opportunity toincrease the rate of spontaneous metastasis of this type of tumor.Metastasis can be assessed at termination of the study by counting thenumber of visible foci per target organ, or measuring the target organweight. The means of these endpoints are compared by Student's t-testafter conducting an F-test, with significance determined at p≦0.05compared to the control group in the experiment.

[0264] 3.3.2. Intraprostatic Assay

[0265] Tumor cells or fragments, of prostatic adenocarcinoma origin, areimplanted directly into a surgically exposed dorsal lobe of the prostatein rodents. The prostate is externalized through an abdominal incisionso that the tumor can be implanted specifically in the dorsal lobe whileverifying that the implant does not enter the seminal vesicles. Thesuccessfully inoculated prostate is replaced in the abdomen and theincisions throught e abdomen and skin are closed. Hormones may also beadministered to the rodents to support the growth of the tumors.Compounds are administered p.o., i.p., i.v., i.m., or s.c. according toa predetermined schedule. Body weights are measured and recorded 2-3times weekly. At a predetermined time, the experiment is terminated andthe animal is dissected. The size of the primary tumor is measured inthree dimensions using either a caliper or an ocular micrometer attachedto a dissecting scope. An F-test is preformed to determine if thevariance is equal or unequal followed by a Student's t-test to comparetumor sizes in the treated and control groups at the end of treatment.Significance is p≦0.05 as compared to the control group. This modelprovides an opportunity to increase the rate of spontaneous metastasisof this type of tumor. Metastasis can be assessed at termination of thestudy by counting the number of visible foci per target organ (i.e., thelungs), or measuring the target organ weight (i.e., the regional lymphnodes). The means of these endpoints are compared by Student's t-testafter conducting an F-test, with significance determined at p≦0.05compared to the control group in the experiment.

[0266] 3.3.3. Intrabronchial Assay

[0267] Tumor cells of pulmonary origin may be implanted intrabronchiallyby making an incision through the skin and exposing the trachea. Thetrachea is pierced with the beveled end of a 25 gauge needle and thetumor cells are inoculated into the main bronchus using a flat-ended 27gauge needle with a 90° bend. Compounds are administered p.o., i.p.,i.v., i.m., or s.c. according to a predetermined schedule. Body weightsare measured and recorded 2-3 times weekly. At a predetermined time, theexperiment is terminated and the animal is dissected. The size of theprimary tumor is measured in three dimensions using either a caliper oran ocular micrometer attached to a dissecting scope. An F-test ispreformed to determine if the variance is equal or unequal followed by aStudent's t-test to compare tumor sizes in the treated and controlgroups at the end of treatment. Significance is p≦0.05 as compared tothe control group. This model provides an opportunity to increase therate of spontaneous metastasis of this type of tumor. Metastasis can beassessed at termination of the study by counting the number of visiblefoci per target organ (i.e., the contralateral lung), or measuring thetarget organ weight. The means of these endpoints are compared byStudent's t-test after conducting an F-test, with significancedetermined at p≦0.05 compared to the control group in the experiment.

[0268] 3.3.4. Intracecal Assay

[0269] Tumor cells of gastrointestinal origin may be implantedintracecally by making an abdominal incision through the skin andexternalizing the intestine. Tumor cells are inoculated into the cecalwall without penetrating the lumen of the intestine using a 27 or 30gauge needle. Compounds are administered p.o., i.p., i.v., i.m., or s.c.according to a predetermined schedule. Body weights are measured andrecorded 2-3 times weekly. At a predetermined time, the experiment isterminated and the animal is dissected. The size of the primary tumor ismeasured in three dimensions using either a caliper or an ocularmicrometer attached to a dissecting scope. An F-test is preformed todetermine if the variance is equal or unequal followed by a Student'st-test to compare tumor sizes in the treated and control groups at theend of treatment. Significance is p≦0.05 as compared to the controlgroup. This model provides an opportunity to increase the rate ofspontaneous metastasis of this type of tumor. Metastasis can be assessedat termination of the study by counting the number of visible foci pertarget organ (i.e., the liver), or measuring the target organ weight.The means of these endpoints are compared by Student's t-test afterconducting an F-test, with significance determined at p≦0.05 compared tothe control group in the experiment.

[0270] 4. Secondary (Metastatic) Antitumor Efficacy

[0271] 4.1. Spontaneous Metastasis

[0272] Tumor cells are inoculated s.c. and the tumors allowed to grow toa predetermined range for spontaneous metastasis studies to the lung orliver. These primary tumors are then excised. Compounds are administeredp.o., i.p., i.v., i.m., or s.c. according to a predetermined schedulewhich may include the period leading up to the excision of the primarytumor to evaluate therapies directed at inhibiting the early stages oftumor metastasis. Observations of morbidity and/or mortality arerecorded daily. Body weights are measured and recorded twice weekly.Potential endpoints include survival time, numbers of visible foci pertarget organ, or target organ weight. When survival time is used as theendpoint the other values are not determined. Survival data is used togenerate Kaplan-Meier curves. Significance is p≦0.05 by a log-rank testcompared to the control group in the experiment. The mean number ofvisible tumor foci, as determined under a dissecting microscope, and themean target organ weights are compared by Student's t-test afterconducting an F-test, with significance determined at p≦0.05 compared tothe control group in the experiment for both of these endpoints.

[0273] 4.2. Forced Metastasis

[0274] Tumor cells are injected into the tail vein, portal vein, or theleft ventricle of the heart in experimental (forced) lung, liver, andbone metastasis studies, respectively. Compounds are administered p.o.,i.p., i.v., i.m., or s.c. according to a predetermined schedule.Observations of morbidity and/or mortality are recorded daily. Bodyweights are measured and recorded twice weekly. Potential endpointsinclude survival time, numbers of visible foci per target organ, ortarget organ weight. When survival time is used as the endpoint theother values are not determined. Survival data is used to generateKaplan-Meier curves. Significance is p≦0.05 by a log-rank test comparedto the control group in the experiment. The mean number of visible tumorfoci, as determined under a dissecting microscope, and the mean targetorgan weights are compared by Student's t-test after conducting anF-test, with significance at p≦0.05 compared to the vehicle controlgroup in the experiment for both endpoints.

EXAMPLE 8 In Vivo Testing of Compounds/Target Validation

[0275] 1. Pain:

[0276] Acute Pain

[0277] Acute pain is measured on a hot plate mainly in rats. Twovariants of hot plate testing are used: In the classical variant animalsare put on a hot surface (52 to 56 □C) and the latency time is measureduntil the animals show nocifensive behavior, such as stepping or footlicking. The other variant is an increasing temperature hot plate wherethe experimental animals are put on a surface of neutral temperature.Subsequently this surface is slowly but constantly heated until theanimals begin to lick a hind paw. The temperature which is reached whenhind paw licking begins is a measure for pain threshold.

[0278] Compounds are tested against a vehicle treated control group.Substance application is performed at different time points viadifferent application routes (i.v., i.p., p.o., i.t., i.c.v., s.c.,intradermal, transdermal) prior to pain testing.

[0279] Persistent Pain

[0280] Persistent pain is measured with the formalin or capsaicin test,mainly in rats. A solution of 1 to 5% formalin or 10 to 100 μg capsaicinis injected into one hind paw of the experimental animal. After formalinor capsaicin application the animals show nocifensive reactions likeflinching, licking and biting of the affected paw. The number ofnocifensive reactions within a time frame of up to 90 minutes is ameasure for intensity of pain.

[0281] Compounds are tested against a vehicle treated control group.Substance application is performed at different time points viadifferent application routes (i.v., i.p., p.o., i.t., i.c.v., s.c.,intradermal, transdermal) prior to formalin or capsaicin administration.

[0282] Neuropathic Pain

[0283] Neuropathic pain is induced by different variants of unilateralsciatic nerve injury mainly in rats. The operation is performed underanesthesia. The first variant of sciatic nerve injury is produced byplacing loosely constrictive ligatures around the common sciatic nerve.The second variant is the tight ligation of about the half of thediameter of the common sciatic nerve. In the next variant, a group ofmodels is used in which tight ligations or transections are made ofeither the L5 and L6 spinal nerves, or the L% spinal nerve only. Thefourth variant involves an axotomy of two of the three terminal branchesof the sciatic nerve (tibial and common peroneal nerves) leaving theremaining sural nerve intact whereas the last variant comprises theaxotomy of only the tibial branch leaving the sural and common nervesuninjured. Control animals are treated with a sham operation.

[0284] Postoperatively, the nerve injured animals develop a chronicmechanical allodynia, cold allodynioa, as well as a thermalhyperalgesia. Mechanical allodynia is measured by means of a pressuretransducer (electronic von Frey Anesthesiometer, IITC Inc.-Life ScienceInstruments, Woodland Hills, SA, USA; Electronic von Frey System,Somedic Sales AB, Hörby, Sweden). Thermal hyperalgesia is. measured bymeans of a radiant heat source (Plantar Test, Ugo Basile, Comerio,Italy), or by means of a cold plate of 5 to 10 □C where the nocifensivereactions of the affected hind paw are counted as a measure of painintensity. A further test for cold induced pain is the counting ofnocifensive reactions, or duration of nocifensive responses afterplantar administration of acetone to the affected hind limb. Chronicpain in general is assessed by registering the circadanian rhytms inactivity (Surjo and Arndt, Universität zu Köln, Cologne, Germany), andby scoring differences in gait (foot print patterns; FOOTPRINTS program,Klapdor et al., 1997. A low cost method to analyse footprint patterns.J. Neurosci. Methods 75, 49-54).

[0285] Compounds are tested against sham operated and vehicle treatedcontrol groups. Substance application is performed at different timepoints via different application routes (i.v., i.p., p.o., i.t., i.c.v.,s.c., intradermal, transdermal) prior to pain testing.

[0286] Inflammatory Pain

[0287] Inflammatory pain is induced mainly in rats by injection of 0.75mg carrageenan or complete Freund's adjuvant into one hind paw. Theanimals develop an edema with mechanical allodynia as well as thermalhyperalgesia. Mechanical allodynia is measured by means of a pressuretransducer (electronic von Frey Anesthesiometer, IITC Inc.-Life ScienceInstruments, Woodland Hills, SA, USA). Thermal hyperalgesia is measuredby means of a radiant heat source (Plantar Test, Ugo Basile, Comerio,Italy, Paw thermal stimulator, G. Ozaki, University of California, USA).For edema measurement two methods are being used. In the first method,the animals are sacrificed and the affected hindpaws sectioned andweighed. The second method comprises differences in paw volume bymeasuring water displacement in a plethysmometer (Ugo Basile, Comerio,Italy).

[0288] Compounds are tested against uninflamed as well as vehicletreated control groups. Substance application is performed at differenttime points via different application routes (i.v., i.p., p.o., i.t.,i.c.v., s.c., intradermal, transdermal) prior to pain testing.

[0289] Diabetic Neuropathic Pain

[0290] Rats treated with a single intraperitoneal injection of 50 to 80mg/kg streptozotocin develop a profound hyperglycemia and mechanicalallodynia within 1 to 3 weeks. Mechanical allodynia is measured by meansof a pressure transducer (electronic von Frey Anesthesiometer, IITCInc.-Life Science Instruments, Woodland Hills, SA, USA).

[0291] Compounds are tested against diabetic and non-diabetic vehicletreated control groups. Substance application is performed at differenttime points via different application. routes (i.v., i.p., p.o., i.t.,i.c.v., s.c., intradermal, transdermal) prior to pain testing.

[0292] 2. Parkinson's Disease

[0293] 6-Hydroxydopamine (6-OH-DA) Lesion

[0294] Degeneration of the dopaminergic nigrostriatal andstriatopallidal pathways is the central pathological event inParkinson's disease. This disorder has been mimicked experimentally inrats using single/sequential unilateral stereotaxic injections of6-OH-DA into the medium forebrain bundle (MFB).

[0295] Male Wistar rats (Harlan Winkelmann, Germany), weighing 200±250 gat the beginning of the experiment, are used. The rats are maintained ina temperature- and humidity-controlled environment under a 12 hlight/dark cycle with free access to food and water when not inexperimental sessions. The following in vivo protocols are approved bythe governmental authorities. All efforts are made to minimize animalsuffering, to reduce the number of animals used, and to utilizealternatives to in vivo techniques.

[0296] Animals are administered pargyline on the day of surgery (Sigma,St. Louis, Mo., USA; 50 mg/kg i.p.) in order to inhibit metabolism of6-OHDA by monoamine oxidase and desmethylimipramine HCl (Sigma; 25 mg/kgi.p.) in order to prevent uptake of 6-OHDA by noradrenergic terminals.Thirty minutes later the rats are anesthetized with sodium pentobarbital(50 mg/kg) and placed in a stereotaxic frame. In order to lesion the DAnigrostriatal pathway 4 μl of 0.01% ascorbic acid-saline containing 8 μgof 6-OHDA HBr (Sigma) are injected into the left medial fore-brainbundle at a rate of 1 μl/min (2.4 mm anterior, 1.49 mm lateral, −2.7 mmventral to Bregma and the skull surface). The needle is left in place anadditional 5 min to allow diffusion to occur.

[0297] Stepping Test

[0298] Forelimb akinesia is assessed three weeks following lesionplacement using a modified stepping test protocol. In brief, the animalsare held by the experimenter with one hand fixing the hindlimbs andslightly raising the hind part above the surface. One paw is touchingthe table, and is then moved slowly sideways (5 s for 1 m), first in theforehand and then in the backhand direction. The number of adjustingsteps is counted for both paws in the backhand and forehand direction ofmovement. The sequence of testing is right paw forehand and backhandadjusting stepping, followed by left paw forehand and backhanddirections. The test is repeated three times on three consecutive days,after an initial training period of three days prior to the firsttesting. Forehand adjusted stepping reveals no consistent differencesbetween lesioned and healthy control animals. Analysis is thereforerestricted to backhand adjusted stepping.

[0299] Balance Test

[0300] Balance adjustments following postural challenge are alsomeasured during the stepping test sessions. The rats are held in thesame position as described in the stepping test and, instead of beingmoved sideways, tilted by the experimenter towards the side of the pawtouching the table. This maneuver results in loss of balance and theability of the rats to regain balance by forelimb movements is scored ona scale ranging from 0 to 3. Score 0 is given for a normal forelimbplacement. When the forelimb movement is delayed but recovery ofpostural balance detected, score 1 is given. Score 2 represents a clear,yet insufficient, forelimb reaction, as evidenced by muscle contraction,but lack of success in recovering balance, and score 3 is given for noreaction of movement. The test is repeated three times a day on eachside for three consecutive days after an initial training period ofthree days prior to the first testing.

[0301] Staircase Test (Paw Reaching)

[0302] A modified version of the staircase test is used for evaluationof paw reaching behavior three weeks following primary and secondarylesion placement. Plexiglass test boxes with a central platform and aremovable staircase on each side are used. The apparatus is designedsuch that only the paw on the same side at each staircase can be used,thus providing a measure of independent forelimb use. For each test theanimals are left in the test boxes for 15 min. The double staircase isfilled with 7×3 chow pellets (Precision food pellets, formula: P,purified rodent diet, size 45 mg; Sandown Scientific) on each side.After each test the number of pellets eaten (successfully retrievedpellets) and the number of pellets taken (touched but dropped) for eachpaw and the success rate (pellets eaten/pellets taken) are countedseparately. After three days of food deprivation (12 g per animal perday) the animals are tested for 11 days. Full analysis is conducted onlyfor the last five days.

[0303] MPTP Treatment

[0304] The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine(MPTP) causes degeneration of mesencephalic dopaminergic (DAergic)neurons in rodents, non-human primates, and humans and, in so doing,reproduces many of the symptoms of Parkinson's disease. MPTP leads to amarked decrease in the levels of dopamine and its metabolites, and inthe number of dopaminergic terminals in the striatum as well as severeloss of the tyrosine hydroxylase (TH)-immunoreactive cell bodies in thesubstantia nigra, pars compacta.

[0305] In order to obtain severe and long-lasting lesions, and to reducemortality, animals receive single injections of MPTP, and are thentested for severity of lesion 7-10 days later. Successive MPTPinjections are administered on days 1, 2 and 3. Animals receiveapplication of 4 mg/kg MPTP hydrochloride (Sigma) in saline once daily.All injections are intraperitoneal (i.p.) and the MPTP stock solution isfrozen between injections. Animals are decapitated on day 11.

[0306] Immunohistology

[0307] At the completion of behavioral experiments, all animals areanaesthetized with 3 ml thiopental (1 g/40 ml i.p., Tyrol Pharma). Themice are perfused transcardially with 0.01 M PBS (pH 7.4) for 2 min,followed by 4% paraformaldehyde (Merck) in PBS for 15 min. The brainsare removed and placed in 4% paraformaldehyde for 24 h at 4° C. Fordehydration they are then transferred to a 20% sucrose (Merck) solutionin 0.1 M PBS at 4° C. until they sink. The brains are frozen inmethylbutan at −20° C. for 2 min and stored at −70° C. Using a sledgemicrotome (mod. 3800-Frigocut, Leica), 25 μm sections are taken from thegenu of the corpus callosum (AP 1.7 mm) to the hippocampus (AP 21.8 mm)and from AP 24.16 to AP 26.72. Forty-six sections are cut and stored inassorters in 0.25 M Tris buffer (pH 7.4) for immunohistochemistry.

[0308] A series of sections is processed for free-floating tyrosinehydroxylase (TH) immunohistochemistry. Following three rinses in 0.1 MPBS, endogenous peroxidase activity is quenched for 10 min in 0.3%H₂O₂±PBS. After rinsing in PBS, sections are preincubated in 10% normalbovine serum (Sigma) for 5 min as blocking agent and transferred toeither primary anti-rat TH rabbit antiserum (dilution 1:2000).

[0309] Following overnight incubation at room temperature, sections forTH immunoreactivity are rinsed in PBS (2×10 min) and incubated inbiotinylated anti-rabbit immunoglobulin G raised in goat (dilution1:200) (Vector) for 90 min, rinsed repeatedly and transferred toVectastain ABC (Vector) solution for 1 h. 3,.3′-Diaminobenzidinetetrahydrochloride (DAB; Sigma) in 0.1 M PBS, supplemented with 0.005%H₂O₂, serves as chromogen in the subsequent visualization reaction.Sections are mounted on to gelatin-coated slides, left to dry overnight,counter-stained with hematoxylin dehydrated in ascending alcoholconcentrations and cleared in butylacetate. Coverslips are mounted onentellan.

[0310] Rotarod Test

[0311] We use a modification of the procedure described by Rozas andLabandeira-Garcia (1997), with a CR-1 Rotamex system (ColumbusInstruments, Columbus, Ohio) comprising an IBM-compatible personalcomputer, a CIO-24 data acquisition card, a control unit, and afour-lane rotarod unit. The rotarod unit consists of a rotating spindle(diameter 7.3 cm) and individual compartments for each mouse. The systemsoftware allows preprogramming of session protocols with varyingrotational speeds (0-80 rpm). Infrared beams are used to detect when amouse has fallen onto the base grid beneath the rotarod. The system logsthe fall as the end of the experiment for that mouse, and the total timeon the rotarod, as well as the time of the fall and all the set-upparameters, are recorded. The system also allows a weak current to bepassed through the base grid, to aid training.

[0312] 3. Dementia

[0313] The Object Recognition Task

[0314] The object recognition task has been designed to assess theeffects of experimental manipulations on the cognitive performance ofrodents. A rat is placed in an open field, in which two identicalobjects are present. The rats inspects both objects during the firsttrial of the object recognition task. In a second trial, after aretention interval of for example 24 hours, one of the two objects usedint the first trial, the ‘familiar’ object, and a novel object areplaced in the open field. The inspection time at each of the objects isregistered. The basic measures in the OR task is the time spent by a ratexploring the two object the second trial. Good retention is reflectedby higher explortation times towards the novel than the ‘familiar’object.

[0315] Administration of the putative cognition enhancer prior to thefirst trial predominantly allows assessment of the effects onacquisition, and eventually on consolidation processes. Administrationof the testing compound after the first trial allows to assess theeffects on consolidation processes, whereas administration before thesecond trial allows to measure effects on retrieval processes.

[0316] The Passive Avoidance Task

[0317] The passive avoidance task assesses memory performance in ratsand mice. The inhibitory avoidance apparatus consists of atwo-compartment box with a light compartment and a dark compartment. Thetwo compartments are separated by a guillotine door that can be operatedby the experimenter. A threshold of 2 cm separates the two compartmentswhen the guillotine door is raised. When the door is open, theillumination in the dark compartment is about 2 lux. The light intensityis about 500 lux at the center of the floor of the light compartment.

[0318] Two habituation sessions, one shock session, and a retentionsession are given, separated by inter-session intervals of 24 hours. Inthe habituation sessions and the retention session the rat is allowed toexplore the apparatus for 300 sec. The rat is placed in the lightcompartment, facing the wall opposite to the guillotine door. After anaccommodation period of 15 sec. the guillotine door is opened so thatall parts of the apparatus can be visited freely. Rats normally avoidbrighly lit areas and will enter the dark compartment within a fewseconds.

[0319] In the shock session the guillotine door between the compartmentsis lowered as soon as the rat has entered the dark compartment with itsfour paws, and a scrambled 1 mA footshock is administered for 2 sec. Therat is removed from the apparatus and put back into its home cage. Theprocedure during the retention session is identical to that of thehabituation sessions.

[0320] The step-through latency, that is the first latency of enteringthe dark compartment (in sec.) during the retention session is an indexof the memory performance of the animal; the longer the latency to enterthe dark compartment, the better the retention is. A testing compound ingiven half an hour before the shock session, together with 1 mg*kg⁻¹scopolamine. Scopolamine impairs the memory performance during theretention session 24 hours later. If the test compound increases theenter latency compared with the scopolamine-treated controls, is islikely to possess cognition enhancing potential.

[0321] The Morris Water Escape Task

[0322] The Morris water escape task measures spatial orientationlearning in rodents. It is a test system that has extensively been usedto investigate the effects of putative therapeutic on the cognitivefunctions of rats and mice. The performance of an animal is assessed ina circular water tank with an escape platform that is submerged about 1cm below the surface of the water. The escape platform is not visiblefor an animal swimming in the water tank. Abundant extra-maze cues areprovided by the furniture in the room, including desks, computerequipment, a second water tank, the presence of the experimenter, and bya radio on a shelf that is playing softly.

[0323] The animals receive four trials during five daily acquisitionsessions. A trial is started by placing an animal into the pool, facingthe wall of the tank. Each of four starting positions in the quadrantsnorth, east, south, and west is used once in a series of four trials;their order is randomized. The escape platform is always in the sameposition. A trial is terminated as soon as the animal had climbs ontothe escape platform or when 90 seconds have elapsed, whichever eventoccurs first. The animal is allowed to stay on the platform for 30seconds. Then it is taken from the platform and the next trial isstarted. If an animal did not find the platform within 90 seconds it isput on the platform by the experimenter and is allowed to stay there for30 seconds. After the fourth trial of the fifth daily session, anadditional trial is given as a probe trial: the platform is removed, andthe time the animal spends in the four quadrants is measured for 30 or60 seconds. In the probe trial, all animals start from the same startposition, opposite to the quadrant where the escape platform had beenpositioned during acquisition.

[0324] Four different measures are taken to evaluate the performance ofan animal during acquisition training: escape latency, traveleddistance, distance to platform, and swimming speed. The followingmeasures are evaluated for the probe trial: time (s) in quadrants andtraveled distance (cm) in the four quadrants. The probe trial providesadditional information about how well an animal learned the position ofthe escape platform. If an animal spends more time and swims a longerdistance in the quadrant where the platform had been positioned duringthe acquisition sessions than in any other quadrant, one concludes thatthe platform position has been learned well.

[0325] In order to assess the effects of putative cognition enhancingcompounds, rats or mice with specific brain lesions that impaircognitive functions, or animals treated with compounds such asscopolamine or MK-801, which interfere with normal learning, or agedanimals which suffer from cognitive deficits, are used.

[0326] The T-Maze Spontaneous Alternation Task

[0327] The T-maze spontaneous alternation task (TeMCAT) assesses thespatial memory performance in mice. The start arm and the two goal armsof the T-maze are provided with guillotine doors which can be operatedmanually by the experimenter. A mouse is put into the start arm at thebeginning of training. The guillotine door is closed. In the firsttrial, the ‘forced trial’, either the left or right goal arm is blockedby lowering the guillotine door. After the mouse has been released fromthe start arm, it will negotiate the maze, eventually enter the opengoal arm, and return to the start position, where it will be confinedfor 5 seconds, by lowering the guillotine door. Then, the animal canchoose freely between the left and right goal arm (all guillotine-doorsopened) diring 14 ‘free choice’ trials. As soon a the mouse has enteredone goal arm, the other one is closed. The mouse eventually returns tothe start arm and is free to visit whichever goalarm it wants afterhaving been confined to the start arm for 5 seconds. After completion of14 free choice trials in one session, the animal is removed from themaze. During training, the animal is never handeled.

[0328] The per-cent alternations out of 14 trials is calculated. Thispercentage and the total time needed to complete the first forced trialand the subsequent 14 free choice trials (in s) is analyzed. Cognitivedeficits are usually induced by an injection of scopolamine, 30 mmbefore the start of the training session. Scopolamine reduced theper-cent alternations to chance level, or below. A cognition enhancer,which is always administered before the training session, will at leastpartially, antagonize the scopolamine-induced reduction in thespontaneous alternation rate.

EXAMPLE 9 Tissue-Specific Expression of Transmembrane Serine Protease

[0329] As a first step to establishing a role for transmembrane serineprotease in the pathogenesis of COPD, expression profiling of the genewas done using real-time quantitative PCR with RNA samples from humanrespiratory tissues and inflammatory cells relevant to COPD. The panelconsisted of total RNA samples lung (adult and fetal), trachea, freshlyisolated alveolar type II cells, cultured human bronchial epithelialcells, cultured small airway epithelial cells, cultured bronchial soothmuscle cells, cultured H441 cells (Clara-like), freshly isolatedneutrophils and monocytes, and cultured monocytes (macrophage-like).Expression of transmembrane serine protease also was evaluated in arange of human tissues using total RNA panels obtained from ClontechLaboratories, UK, Ltd. The tissues were adrenal gland, bone marrow,brain, colon, heart, kidney, liver, lung, mammary gland, pancreas,prostate, salivary gland, skeletal muscle, small intestine, spleen,stomach, testis, thymus, trachea, thyroid, and uterus.

[0330] Real-time quantitative PCR. Expression profiling of the targetgene was performed using real-time quantitative PCR, a development ofthe kinetic analysis of PCR first described in Higuchi et al.,BioTechnology 10, 413-17, 1992, and Higuchi et al., BioTechnology 11,1026-30, 1993. The principle is that at any given cycle within theexponential phase of. PCR, the amount of product is proportional to theinitial number of template copies.

[0331] PCR amplification is performed in the presence of anoligonucleotide probe (TaqMan probe) that is complementary to the targetsequence and labeled with a fluorescent reporter dye and a quencher dye.During the extension phase of PCR, the probe is cleaved by the 5′-3′endonuclease activity of Taq DNA polymerase, releasing the fluorophorefrom the effect of the quenching dye (Holland et al., Proc. Natl. Acad.Sci. U.S.A. 88, 7276-80, 1991). Because the fluorescence emissionincreases in direct proportion to the amount of the specific amplifiedproduct, the exponential growth phase of PCR product can be detected andused to determine the initial template concentration (Heid et al.,Genome Res. 6, 986-94, 1996, and Gibson et al., Genome Res. 6, 995-1001,1996).

[0332] Real-time quantitative PCR was done using an ABI Prism 7700Sequence Detector. The C_(T) value generated for each reaction was usedto determine the initial template concentration (copy number) byinterpolation from a universal standard curve. The level of expressionof the target gene in each sample was calculated relative to the samplewith the lowest expression of the gene.

[0333] RNA extraction and cDNA preparation. Total RNA from each of therespiratory tissues and inflammatory cell types listed above wereisolated using Qiagen's RNeasy system according to the manufacturer'sprotocol (Crawley, West Sussex, UK). The concentration of purified RNAwas determined using a RiboGreen RNA quantitation kit (Molecular ProbesEurope, The Netherlands). For the preparation of cDNA, 1 μg of total RNAwas reverse transcribed in a final volume of 20 μl, using 200 U ofSUPERSCRIPT™ RNase H⁻ Reverse Transcriptase (Life Technologies, Paisley,UK), 10 mM dithiothreitol, 0.5 mM of each dNTP and 5 μM random hexamers(Applied Biosystems, Warrington, Cheshire, UK) according to themanufacturer's protocol.

[0334] TaqMan quantitative analysis. Specific primers and probe weredesigned according to the recommendations of PE Applied Biosystems. Theprobe was labeled at the 5′ end with FAM (6-carboxyfluorescein).Quantification PCR was performed with 5 ng of reverse transcribed RNAfrom each sample. Each determination is done in duplicate.

[0335] The assay reaction mix was as follows: 1× final TaqMan UniversalPCR Master Mix (from 2× stock) (PE Applied Biosystems, California); 900nM forward primer; 900 nM reverse primer; 200 nM probe; 5 ng cDNA; andwater to 25 μl.

[0336] Each of the following steps were carried out once: pre PCR, 2minutes at 50° C., and 10 minutes at 95° C. The following steps arecarried out 40 times: denaturation, 15 seconds at 95° C.,annealing/extension, 1 minute at 60° C.

[0337] All experiments were performed using an ABI Prism 7700 SequenceDetector (PE Applied Biosystems, California). At the end of the run,fluorescence data acquired during PCR were processed as described in theABI Prism 7700 user's manual to achieve better background subtraction aswell as signal linearity with the starting target quantity.

[0338] Tables 1 and 2 show the results of expression profiling fortransmembrane serine protease using the indicated cell and tissuesamples. For Table 1, the cells are defined as follows: HBEC, culturedhuman bronchial epithelial cells; H441, a Clara-like cell line; SAE,cultured small airway epithelial cells; SMC, cultured airway smoothmuscle cells; All, freshly isolated human alveolar type II cells; Neut,freshly isolated circulating neutrophils; Mono, freshly isolatedmonocytes; and CM, cultured monocytes. Other letters identify the donor.The results are shown graphically in FIGS. 2 and 3. TABLE 1 TissueRelative expression Lung 2200.562359 Trachea 258.4435393 HBEC 1513.406875 HBEC 2 780.9633981 H441 5636.839641 SMC 0 SAE 4154.786196 AII1428.383024 Fetal lung 59.1577544 COPD Neut 1 2.529186063 COPD Neut 2 1COPD Neut 4 8.733909349 GAP Neut 5.889527853 AEM Neut 0 AT Neut2.529186063 KN Neut 0 SM Mono 31.93586472 DLF Mono 26.90014866 DS Mono110.9856117 RLH CM 59.91460217 CTP CM 158.4289996

[0339] TABLE 2 Tissue Relative expression Adrenal gland 13.45548325 BoneMarrow 18.60661194 Brain 5.186534845 Colon 43.05318645 Heart 6.118448375HL60 1 Kidney 23.68930394 Liver 1.705502263 Lung 144.941683 Mammarygland 128.454789 Pancreas 27.59276994 Prostate 32.75814493 Salivarygland 129.273736 Skeletal Muscle 35.57961956 Sm Intest 20.08113704Spleen 37.91423521 Stomach 13.03461776 Testis 32.34441495 Thymus43.05318645 Thyroid 295.3447804 Uterus 86.62062241

EXAMPLE 10 Expression of Human Transmembrane Serine Protease in Normaland Cancer Tissues

[0340] RNA Extraction and cDNA Preparation

[0341] Total RNA used for Taqman quantitative analysis were eitherpurchased (Clontech, California) or extracted from tissues using TRIzolreagent (Life Technologies, Maryland) according to a modified vendorprotocol which utilizes the RNeasy protocol (Qiagen, California) Fiftyμg of each RNA were treated with DNase I using RNase free- DNase(Qiagen, California) for use with RNeasy or QiaAmp columns.

[0342] After elution and quantitation with Ribogreen (Molecular ProbesInc., OR) each sample was reverse transcribed using the GibcoBRLSuperscript II First Strand Synthesis System for RT-PCR according tovendor protocol (Life Technologies, Maryland). The final concentrationof RNA in the reaction mix was 50 ng/μL. Reverse transcription wasperformed with 0.5 ug of Oligo dT primer.

[0343] TaqMan Quantitative Analysis

[0344] Specific primers and probe were designed according to PE AppliedBiosystems recommendations and are listed below: forward primer:5′-(CTGCCAGCAGCTGGGTTTC)-3′ (SEQ ID NO:9) reverse primer:5′-(AGGCTTTCCTGGATGGTGGA)-3′ (SEQ ID NO:10) probe:5′-(FAM)-(CAACCTCGGTTGTCCGGTGAGCACTCT) (TAMRA)-3′ (SEQ ID NO:13)

[0345] where

[0346] FAM=6-carboxy-fluorescein

[0347] and TAMRA=6-carboxy-tetramethyl-rhodamine.

[0348] The expected length of the PCR product was −111 bp.

[0349] Quantitation experiments were performed on 25 ng of reversetranscribed RNA from each sample. Each determination was done induplicate. 18S ribosomal RNA was measured as a control using thePre-Developed TaqMan Assay Reagents (PDAR)(PE Applied Biosystems,California). Assay reaction mix was as follows: final TaqMan UniversalPCR Master Mix (2×) 1× (PE Applied Biosystems, CA) PDAR control-18S RNA(20×) 1× Forward primer 300 nM Reverse primer 300 nM Probe 200 nM cDNA25 ng Water to 25 uL

[0350] PCR conditions: Once:  2’ minutes at 50° C.  10 minutes at 95° C.40 cycles: 15 sec. at 95° C.   1 minute at 60° C.

[0351] The experiment was performed on an ABI Prism 7700 SequenceDetector (PE Applied Biosystems, California). At the end of the run,fluorescence data acquired during PCR were processed as described in theABI Prism 7700 user's manual. Fold change was calculated using thedelta-delta CT method with normalization to the 18S values and copynumber conversion was performed without normalization using the formulaCn=10^((Ct-40.007)/−3.623). The results are shown in FIGS. 7 and 8.

EXAMPLE 11 Northern Analysis

[0352] Northern analysis was done using a human 12-lane MTN purchasedfrom Clontech (California). The entire coding sequence of transmembraneserine protease was used as a probe and labeled with ³²P using theRediprime II labelling system (Amersham Pharmacia Biotech, N.J.).Hybridization and washing conditions were performed according to theNorthern Max kit from Ambion (TX). The blot was exposed for 16 hours andthe Storm 860 phosphoimager (Amersham Pharmacia Biotech, N.J.) was usedto visualize the Northern analysis. The results are shown in FIG. 6.

EXAMPLE 12 Cloning of Full-Length Human Transmembrane Serine Protease

[0353] The human EST sequence having accession number BE732381 (SEQ IDNO:128) was found to overlap with the 5′ end of the sequence shown inSEQ ID NO:35. BE732381 was used to search the public databases foroevrlapping EST and genomic sequences. No human ESTs or genomicsequences were found to extend the 5′ end of that sequence. However, 5overlapping mouse EST sequences were identified that were 80-90%identical to the sequence shown in SEQ ID NO:35 at the nucleotide level.The accession numbers for these EST sequences are: BE285038 (SEQ IDNO:127), BE289529 (SEQ ID NO:128), BE290038 (SEQ ID NO 29), BE309103(SEQ ID NO:30), and BE286322 SEQ ID NO:31). Four of the mouse ESTsequences overlapped with the 5′ end of SEQ ID NO:35 and significantlyextended the sequence. Translation of these ESTs revealed a putativetransmembrane domain, indicating that this protein is a transmembraneserine protease.

[0354] One of the mouse ESTs, BE289529, was then selected to search thehuman genomic and EST databases. No significantly overlapping human ESTsequences were identified. However, BE289529 aligned with a humangenomic entry, AP000757. AP000757 is an unordered genomic entry forhuman chromosome 11. The AP000757 exon significantly extended thepredicted sequence of human transmembrane protease but did not appear toencode an appropriate translational start codon. The AP000757 exon wasthen used to search the public databases for overlapping EST sequences.

[0355] A single human EST, BE280394 (SEQ ID NO:34), was identified thatfurther extended the sequence at the 5′ end. BE280394 was found tocontain an in-frame translational start codon. This putative start codonand its flanking sequences resemble the Kozak consensus translationalstart sequence, suggesting that this is the translational start site forthe transmembrane serine protease protein. Oligonucleotide primersflanking the predicted coding sequence were then designed to confirmthat the predicted full length sequence was expressed.

[0356] PCR products of the appropriate size were identified in cDNApools generated from placenta and spleen poly A+ RNA (Clontech),confirming expression of the predicted full length cDNA. The PCRproducts were cloned into the pCRII vector (Invitrogen) and sequenced.The nucleotide and amino acid sequences are shown in SEQ ID NOS:11 and12, respectively.

[0357] One of the clones isolated was a putative splice variant thatlacked a region that encodes the putative transmembrane domain.

[0358] A plasmid containing a cDNA encoding the human transmembraneserine protease, pCRII-TMSP3, was deposited under the provisions of theBudapest Treaty with the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209 on Jun. 5, 2001, andassigned Accession No. ______. A restriction map of the depositedplasmid is shown in FIG. 9.

REFERENCES

[0359] 1. Nicolson (1988) Organ specificity of tumor metastasis: Role ofpreferential adhesion, invasion and growth of malignant cells atspecific secondary sites. Cancer Met. Rev. 7, 143-188.

[0360] 2. Liotta et al. (1983) Tumor invasion and the extracellularmatrix. Lab. Invest. 49, 639-649.

[0361] 3. Vlodavsky et al. (1987) Endothelial cell-derived basicfibroblast growth factor: Synthesis and deposition into subendothelialextracellular matrix. Proc. Natl. Acad. Sci. USA 84, 2292-2296.

[0362] 4. Folkman et al. (1980) A heparin-binding angiogenicprotein—basic fibroblast growth factor—is stored within basementmembrane. Am. J. Pathol. 130, 393400.

[0363] 5. Cardon-Cardo et al. (1990) Expression of basic fibroblastgrowth factor in normal human tissues. Lab. Invest. 63, 832-840.

[0364] 6. Vlodavsky et al. (1991) Extracellular sequestration andrelease of fibroblast growth factor: a regulatory mechanism? TrendsBiochem. Sci. 16, 268-271.

[0365] 7. Vlodavsky et al. (1993) Extracellular matrix-bound growthfactors, enzymes and plasma proteins. In BASEMENT MEMBRANES: CELLULARAND MOLECULAR ASPECTS Rohrbach & Timpl, eds., pp327-343. Academic PressInc., Orlando, Fla.

[0366] 8. Ross (1993) The pathogenesis of atherosclerosis: a perspectivefor the 1990s. Nature

[0367] (Lond.). 362, 801-809.

[0368] 9. McCarthy et al. (1986). Human fibronectin contains distinctadhesion- and motility-promoting domains for metastatic melanoma cells.J. Cell Biol. 102, 179-88.

[0369] 10. Van Muijen et al. (1995) Properties of metastasizing andnon-metastasizing human melanoma cells. Recent Results in CancerResearch 139, 104-22.

[0370] 11. Price et al. (1997) The Biochemistry of Cancer Dissemination,in Critical Reviews in Biochemistry and Mol. Biol. 32, 175-253.

[0371] 12. Leytus et al. A novel trypsin-like serine protease (hepsin)with a putative transmembrane domain expressed by human liver andhepatoma cells. Biochemistry. 1988 February 9;27(3): 1067-74.

[0372] 13. Tanimoto et al. Hepsin, a cell surface serine proteaseidentified in hepatoma cells, is overexpressed in ovarian cancer. CancerRes. 1997 July 15;57(14):2884-7.

[0373] 14. Zacharski et al., Expression of the factor VII activatingprotease, hepsin, in situ in renal cell carcinoma. Thromb Haemost. 1998April;79(4):876-7.

1 36 1 402 DNA Homo sapiens misc_feature (1)...(402) n = A,T,C or G 1aatgcccttc ccagcggtat atctccctcc agtgttccca ctgcggactg agggccatga 60ccgggcggat cgtgggaggg gcgctggcct cggatagcaa gtggccttgg caagtgagcc 120tgcacttcgg caccacccac atctgtggag gcacgctcat tgacgcccag tgggtgctca 180ctnccgccca ctgcttcttc gtgnacccgg gagaaggtcc tggagggctg gaaggtgtac 240cgggcacca gcaacctgca ccagttgcct gaggcagcct ccattgccga gatcatcatc 300acagcaatt acaccgatga ggaggacgac tatgacatcg ccctcatgcg gctgttcaag 360cccttgacc ctgttccggt gagggaattt tgcatttccc gt 402 2 285 DNA Homo sapiens2 ccatgaccgg gcggatcgtg ggaggggcgc tggcctcgga tagcaagtgg ccttggcaag 60tgagtctgca cttcggcacc acccacatct gtggaggcac gctcattgac gcccagtggg 120tgctcactgc cgcccactgc ttcttcgtga cccgggagaa ggtcctggag ggctggaagg 180tgtacgcggg caccagcaac ctgcaccagt tgcctgaggc agcctccatt gccgagatca 240tcatcaacag caattacacc gatgaggagg acgactatga catcg 285 3 600 DNA Homosapiens misc_feature (1)...(600) n = A,T,C or G 3 gagggctgga aggtgtacgcgggcaccagc aacctgcacc agttgcctga ggcagcctcc 60 attgccgaga tcatcatcaacagcaattac accgatgagg aggacgacta tgacatcgcc 120 ctcatgcggc tgtccaagcccctgaccctg tccggtgagg gaatctgcac tccccgctct 180 cctgcccccc agccccagcaccctctgcag ccctcgcact tgtcagcatc tgtcaactca 240 tatccgggcc ccaaagcttctgcagggcag aagtcaaaga ctcttaaaga tccttacatg 300 gaacacttct gttttataattagggaaact gaagcccaag ggttataaat aagtttgctc 360 caaatgacac atctcacattacaaattgat gacggagtca gggcttgggt actgatctta 420 atcaatagat tgaattctttcactggtatt aactgagcac ctaggggcca aacgctatgg 480 aggcatttc acacatatgatttcatttac tcttcacaac caaccctgtg gagcaggcac 540 attattaac ttcatttgacatatgangaa atggagcttt acagagagat aattacctga 600 4 591 DNA Homo sapiensmisc_feature (1)...(591) n = A,T,C or G 4 gagggctgga aggtgtacgcgggcaccagc aacctgcacc agttgcctga ggcagcctcc 60 attgccgaga tcatcatcaacagcaattac accgatgagg aggacgacta tgacatcgcc 120 ctcatgcggc tgtccaagcccctgaccctg tccggtgagg gaatctgcac tccccgctct 180 cctgcccccc agccccagcaccctctgcag ccctcgcact tgtcagcatc tgtcaactca 240 tatccgggcc ccaaagcttctgcagggcag aagtcaaaga ctcttaaaga tccttacatg 300 gaacacttct gttttataattagggaaact gaagcccaag ggttataaat aagtttgctc 360 caaatgacac atctcacattacaaattgat gacggagtca gggcttgggt actgatctta 420 atcaatagat tgaattctttcactggtatt aactgagcac ctaggggcca aacgctatgg 480 aggcatttc acacatatgatttcatttac tcttcacaac caaccctgtg gagcangcac 540 attattaac ttcatttgacatatgangaa atggagcttt acagagagat a 591 5 286 DNA Homo sapiens 5gcgatgtcat agtcgtcctc ctcatcggcg taattgctgt tgatgatgat ctcggcaatg 60gaggctgcct caggcaactg gtgcaggttg ctggtgcccg cgtacacctt ccagccctcc 120aagaccttct cccgggtcac gaagaagcag tgggcggcag tgagcaccca ctgggcgtca 180atgagcgtgc ctccacagat gtgggtggtg ccgaagtgct gactcacttg ccaaggccac 240ttgctattcg aggccagcgc cccttccacg attcgcccgg tcatgg 286 6 384 DNA Homosapiens 6 gagggctgga aggtgtacgc gggcaccagc aacctgcacc agttgcctgaggagcctcca 60 ttgccgagat catcatcaac agcaattaca ccgatgagga ggacgactatgacatcgccc 120 tcatgcggct gtccaagccc ctgaccctgt ccggtgaggg aatctgcactccccgctctc 180 ctgcccccca gccccagcac cctctgcagc cctcgcactt gtcagcatctgtcaactcat 240 tccgggccc caaagcttct gcagggcaga agtcaaagac tcttaaagatccttacatgg 300 acacttctg ttttataatt agggaaactg aagcccaagg gttataaataagtttgctcc 360 aatgacaca tctcacatta caaa 384 7 471 DNA Homo sapiensmisc_feature (1)...(471) n = A,T,C or G 7 tttttttttt ntttttttttttggagcaaa cttatttana acccttgggc ttcagttncc 60 ctaattataa aacagaagtntnccatgtaa ggnncttnaa gagtctttga cttctgccct 120 gcagaagctt tggggcccggatatgagttg acagatgctg acaagtgcga gggctgcaga 180 gggtnctggg gctggggggcaggagagcgg ggagtgcaga ttccctcacc ggacagggtc 240 aggggnttgg acagccgcatgagggcgatg tcatagtcgt cctcctcatc ggtgtaatnn 300 ctnttgatga tgatctcggcaatggaggct gcctcaggca actgggtnca ggttnctggg 360 tncccncgta acaccttccagccntccagg nccttttccc gggtcacgaa gaagcagtng 420 ggccgcaatt agcacccactgggggtcaat gaggctgccn ccacanattt g 471 8 235 DNA Homo sapiens 8gggctggaag gtgtacgcgg gcaccagcaa cctgcaccag ttgcctgagc agcctccatt 60gccgagatca tcatcaacag caattacacc gatgaggagg acgactatga catcgccctc 120atgcggctgt ccaagcccct gaccctgtcc ggtgagggaa tctgcactcc ccgctctcct 180gccccccagc cccagcaccc tctgcagccc tcgcacttgt cagcatctgt caact 235 9 19DNA Homo sapiens 9 ctgccagcag ctgggtttc 19 10 20 DNA Homo sapiens 10aggctttcct ggatggtgga 20 11 1748 DNA Homo sapiens 11 ctcagagaccatggagaggg acagccacgg gaatgcatct ccagcaagaa caccttcagc 60 tggagcatctccagcccagg catctccagc tgggacacct ccaggccggg catctccagc 120 ccaggcatctccagcccagg catctccagc tgggacacct ccgggccggg catctccagc 180 ccaggcatctccagctggta cacctccagg ccgggcatct ccaggccggg catctccagc 240 ccaggcatctccagcccggg catctccggc tctggcatca ctttccaggt cctcatccgg 300 caggtcatcatccgccaggt cagcctcggt gacaacctcc ccaaccagag tgtaccttgt 360 tagagcaacaccagtggggg ctgtacccat ccgatcatct cctgccaggt cagcaccagc 420 aaccagggccaccagggaga gcccaggtac gagcctgccc aagttcacct ggcgggaggg 480 ccagaagcagctaccgctca tcgggtgcgt gctcctcctc attgccctgg tggtttcgct 540 catcatcctcttccagttct ggcagggcca cacagggatc aggtacaagg agcagaggga 600 gagctgtcccaagcacgctg ttcgctgtga cggggtggtg gactgcaagc tgaagagtga 660 cgagctgggctgcgtgaggt ttgactggga caagtctctg cttaaaatct actctgggtc 720 ctcccatcagtggcttccca tctgtagcag caactggaat gactcctact cagagaagac 780 ctgccagcagctgggtttcg agagtgctca ccggacaacc gaggttgccc acagggattt 840 tgccaacagcttctcaatct tgagatacaa ctccaccatc caggaaagcc tccacaggtc 900 tgaatgcccttcccagcggt atatctccct ccagtgttcc cactgcggac tgagggccat 960 gaccgggcggatcgtgggag gggcgctggc ctcggatagc aagtggcctt ggcaagtgag 1020 tctgcacttcggcaccaccc acatctgtgg aggcacgctc attgacgccc agtgggtgct 1080 cactgccgcccactgcttct tcgtgacccg ggagaaggtc ctggagggct ggaaggtgta 1140 cgcgggcaccagcaacctgc accagttgcc tgaggcagcc tccattgccg agatcatcat 1200 caacagcaattacaccgatg aggaggacga ctatgacatc gccctcatgc ggctgtccaa 1260 gcccctgaccctgtccgctc acatccaccc tgcttgcctc cccatgcatg gacagacctt 1320 tagcctcaatgagacctgct ggatcacagg ctttggcaag accagggaga cagatgacaa 1380 gacatcccccttcctccggg aggtgcaggt caatctcatc gacttcaaga aatgcaatga 1440 ctacttggtctatgacagtt accttacccc aaggatgatg tgtgctgggg accttcgtgg 1500 gggcagagactcctgccagg gagacagcgg ggggcctctt gtctgtgagc agaacaaccg 1560 ctggtacctggcaggtgtca ccagctgggg cacaggctgt ggccagagaa acaaacctgg 1620 tgtgtacaccaaagtgacag aagttcttcc ctggatttac agcaagatgg agagcgaggt 1680 cgattcagaaaatcctaac cagctggcct gctgctctgc acagcaccgg ctgctgtgac 1740 cgagaaa 174812 562 PRT Homo sapiens 12 Met Glu Arg Asp Ser His Gly Asn Ala Ser ProAla Arg Thr Pro Ser 1 5 10 15 Ala Gly Ala Ser Pro Ala Gln Ala Ser ProAla Gly Thr Pro Pro Gly 20 25 30 Arg Ala Ser Pro Ala Gln Ala Ser Pro AlaGln Ala Ser Pro Ala Gly 35 40 45 Thr Pro Pro Gly Arg Ala Ser Pro Ala GlnAla Ser Pro Ala Gly Thr 50 55 60 Pro Pro Gly Arg Ala Ser Pro Gly Arg AlaSer Pro Ala Gln Ala Ser 65 70 75 80 Pro Ala Arg Ala Ser Pro Ala Leu AlaSer Leu Ser Arg Ser Ser Ser 85 90 95 Gly Arg Ser Ser Ser Ala Arg Ser AlaSer Val Thr Thr Ser Pro Thr 100 105 110 Arg Val Tyr Leu Val Arg Ala ThrPro Val Gly Ala Val Pro Ile Arg 115 120 125 Ser Ser Pro Ala Arg Ser AlaPro Ala Thr Arg Ala Thr Arg Glu Ser 130 135 140 Pro Gly Thr Ser Leu ProLys Phe Thr Trp Arg Glu Gly Gln Lys Gln 145 150 155 160 Leu Pro Leu IleGly Cys Val Leu Leu Leu Ile Ala Leu Val Val Ser 165 170 175 Leu Ile IleLeu Phe Gln Phe Trp Gln Gly His Thr Gly Ile Arg Tyr 180 185 190 Lys GluGln Arg Glu Ser Cys Pro Lys His Ala Val Arg Cys Asp Gly 195 200 205 ValVal Asp Cys Lys Leu Lys Ser Asp Glu Leu Gly Cys Val Arg Phe 210 215 220Asp Trp Asp Lys Ser Leu Leu Lys Ile Tyr Ser Gly Ser Ser His Gln 225 230235 240 Trp Leu Pro Ile Cys Ser Ser Asn Trp Asn Asp Ser Tyr Ser Glu Lys245 250 255 Thr Cys Gln Gln Leu Gly Phe Glu Ser Ala His Arg Thr Thr GluVal 260 265 270 Ala His Arg Asp Phe Ala Asn Ser Phe Ser Ile Leu Arg TyrAsn Ser 275 280 285 Thr Ile Gln Glu Ser Leu His Arg Ser Glu Cys Pro SerGln Arg Tyr 290 295 300 Ile Ser Leu Gln Cys Ser His Cys Gly Leu Arg AlaMet Thr Gly Arg 305 310 315 320 Ile Val Gly Gly Ala Leu Ala Ser Asp SerLys Trp Pro Trp Gln Val 325 330 335 Ser Leu His Phe Gly Thr Thr His IleCys Gly Gly Thr Leu Ile Asp 340 345 350 Ala Gln Trp Val Leu Thr Ala AlaHis Cys Phe Phe Val Thr Arg Glu 355 360 365 Lys Val Leu Glu Gly Trp LysVal Tyr Ala Gly Thr Ser Asn Leu His 370 375 380 Gln Leu Pro Glu Ala AlaSer Ile Ala Glu Ile Ile Ile Asn Ser Asn 385 390 395 400 Tyr Thr Asp GluGlu Asp Asp Tyr Asp Ile Ala Leu Met Arg Leu Ser 405 410 415 Lys Pro LeuThr Leu Ser Ala His Ile His Pro Ala Cys Leu Pro Met 420 425 430 His GlyGln Thr Phe Ser Leu Asn Glu Thr Cys Trp Ile Thr Gly Phe 435 440 445 GlyLys Thr Arg Glu Thr Asp Asp Lys Thr Ser Pro Phe Leu Arg Glu 450 455 460Val Gln Val Asn Leu Ile Asp Phe Lys Lys Cys Asn Asp Tyr Leu Val 465 470475 480 Tyr Asp Ser Tyr Leu Thr Pro Arg Met Met Cys Ala Gly Asp Leu Arg485 490 495 Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu ValCys 500 505 510 Glu Gln Asn Asn Arg Trp Tyr Leu Ala Gly Val Thr Ser TrpGly Thr 515 520 525 Gly Cys Gly Gln Arg Asn Lys Pro Gly Val Tyr Thr LysVal Thr Glu 530 535 540 Val Leu Pro Trp Ile Tyr Ser Lys Met Glu Ser GluVal Arg Phe Arg 545 550 555 560 Lys Ser 13 27 DNA Homo sapiens 13caacctcggt tgtccggtga gcactct 27 14 492 PRT Homo sapiens 14 Met Ala LeuAsn Ser Gly Ser Pro Pro Ala Ile Gly Pro Tyr Tyr Glu 1 5 10 15 Asn HisGly Tyr Gln Pro Glu Asn Pro Tyr Pro Ala Gln Pro Thr Val 20 25 30 Val ProThr Val Tyr Glu Val His Pro Ala Gln Tyr Tyr Pro Ser Pro 35 40 45 Val ProGln Tyr Ala Pro Arg Val Leu Thr Gln Ala Ser Asn Pro Val 50 55 60 Val CysThr Gln Pro Lys Ser Pro Ser Gly Thr Val Cys Thr Ser Lys 65 70 75 80 ThrLys Lys Ala Leu Cys Ile Thr Leu Thr Leu Gly Thr Phe Leu Val 85 90 95 GlyAla Ala Leu Ala Ala Gly Leu Leu Trp Lys Phe Met Gly Ser Lys 100 105 110Cys Ser Asn Ser Gly Ile Glu Cys Asp Ser Ser Gly Thr Cys Ile Asn 115 120125 Pro Ser Asn Trp Cys Asp Gly Val Ser His Cys Pro Gly Gly Glu Asp 130135 140 Glu Asn Arg Cys Val Arg Leu Tyr Gly Pro Asn Phe Ile Leu Gln Met145 150 155 160 Tyr Ser Ser Gln Arg Lys Ser Trp His Pro Val Cys Gln AspAsp Trp 165 170 175 Asn Glu Asn Tyr Gly Arg Ala Ala Cys Arg Asp Met GlyTyr Lys Asn 180 185 190 Asn Phe Tyr Ser Ser Gln Gly Ile Val Asp Asp SerGly Ser Thr Ser 195 200 205 Phe Met Lys Leu Asn Thr Ser Ala Gly Asn ValAsp Ile Tyr Lys Lys 210 215 220 Leu Tyr His Ser Asp Ala Cys Ser Ser LysAla Val Val Ser Leu Arg 225 230 235 240 Cys Leu Ala Cys Gly Val Asn LeuAsn Ser Ser Arg Gln Ser Arg Ile 245 250 255 Val Gly Gly Glu Ser Ala LeuPro Gly Ala Trp Pro Trp Gln Val Ser 260 265 270 Leu His Val Gln Asn ValHis Val Cys Gly Gly Ser Ile Ile Thr Pro 275 280 285 Glu Trp Ile Val ThrAla Ala His Cys Val Glu Lys Pro Leu Asn Asn 290 295 300 Pro Trp His TrpThr Ala Phe Ala Gly Ile Leu Arg Gln Ser Phe Met 305 310 315 320 Phe TyrGly Ala Gly Tyr Gln Val Gln Lys Val Ile Ser His Pro Asn 325 330 335 TyrAsp Ser Lys Thr Lys Asn Asn Asp Ile Ala Leu Met Lys Leu Gln 340 345 350Lys Pro Leu Thr Phe Asn Asp Leu Val Lys Pro Val Cys Leu Pro Asn 355 360365 Pro Gly Met Met Leu Gln Pro Glu Gln Leu Cys Trp Ile Ser Gly Trp 370375 380 Gly Ala Thr Glu Glu Lys Gly Lys Thr Ser Glu Val Leu Asn Ala Ala385 390 395 400 Lys Val Leu Leu Ile Glu Thr Gln Arg Cys Asn Ser Arg TyrVal Tyr 405 410 415 Asp Asn Leu Ile Thr Pro Ala Met Ile Cys Ala Gly PheLeu Gln Gly 420 425 430 Asn Val Asp Ser Cys Gln Gly Asp Ser Gly Gly ProLeu Val Thr Ser 435 440 445 Asn Asn Asn Ile Trp Trp Leu Ile Gly Asp ThrSer Trp Gly Ser Gly 450 455 460 Cys Ala Lys Ala Tyr Arg Pro Gly Val TyrGly Asn Val Met Val Phe 465 470 475 480 Thr Asp Trp Ile Tyr Arg Gln MetLys Ala Asn Gly 485 490 15 23 PRT Artificial Sequence BLOCKS BL00495 15Ala Gly Gly Gly Asp Cys Gly Asp Ser Gly Gly Pro Leu Val Cys Asn 1 5 1015 Arg Trp Leu Gly Thr Ser Trp 20 16 12 PRT Artificial Sequence BLOCKSBL1253G 16 Asp Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys 1 5 10 17 17PRT Artificial Sequence BLOCKS BL00134A 17 Cys Gly Gly Thr Leu Ile AspAla Gln Trp Val Leu Thr Ala Ala His 1 5 10 15 Cys 18 38 PRT ArtificialSequence BLOCKS BL00021D 18 Gly Pro Leu Val Cys Glu Gln Asn Asn Arg TrpTyr Leu Gly Val Thr 1 5 10 15 Ser Trp Gly Gly Cys Gly Gln Arg Asn LysPro Gly Val Tyr Thr Lys 20 25 30 Val Thr Leu Pro Trp Ile 35 19 24 PRTArtificial Sequence BLOCKS BL01243H 19 Tyr Leu Gly Ser Trp Gly Gly CysGly Gln Arg Asn Lys Pro Gly Val 1 5 10 15 Tyr Thr Lys Val Thr Leu TrpIle 20 20 17 PRT Artificial Sequence BLOCKS BL00021B 20 Cys Gly Gly ThrLeu Ile Asp Gln Trp Val Leu Thr Ala Ala His Cys 1 5 10 15 Phe 21 18 PRTArtificial Sequence BLOCKS BL00495O 21 Gly Gly Cys Gly Gln Arg Pro GlyVal Tyr Thr Lys Val Glu Trp Ile 1 5 10 15 Lys Ala 22 23 PRT ArtificialSequence BLOCKS BL00134B 22 Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro LeuVal Cys Glu Asn Asn 1 5 10 15 Arg Trp Tyr Leu Ala Gly Val 20 23 12 PRTArtificial Sequence BLOCKS BL01209 23 Cys Asp Gly Val Val Asp Cys LysLys Ser Asp Glu 1 5 10 24 20 PRT Artificial Sequence BLOCKS BL01253F 24Ala Ser Phe Leu Arg Glu Gln Val Leu Lys Cys Val Tyr Ser Thr Pro 1 5 1015 Met Cys Ala Gly 20 25 17 PRT Artificial Sequence BLOCKS BL00495L 25Ser Ser Ile Glu Ile Ile Ile Asn Tyr Glu Tyr Asp Ile Ala Leu Leu 1 5 1015 Pro 26 14 PRT Artificial Sequence BLOCKS BL00134C 26 Pro Gly Val TyrThr Lys Val Thr Glu Val Leu Pro Trp Ile 1 5 10 27 10 PRT ArtificialSequence BLOCKS BL01253D 27 Cys Gly Gly Leu Ile Trp Val Leu Thr Ala 1 510 28 834 DNA Homo sapiens 28 gctgggctgc gtgaggtttg actgggacaagtctctgctt aaaatctact ctgggtcctc 60 ccatcagtgg cttcccatct gtagcagcaactggaatgac tcctactcag agaagacctg 120 ccagcagctg ggtttcgaga gtgctcaccggacaaccgag gttgcccaca gggattttgc 180 caacagcttc tcaatcttga gatacaactccaccatccag gaaagcctcc acaggtctga 240 atgcccttcc cagcggtata tctccctccagtgttcccac tgcggactga gggccatgac 300 cgggcggatc gtgggagggg cgctggcctcggatagcaag tggccttggc aagtgagtct 360 gcacttcggc accacccaca tctgtggaggcacgctcatt gacgcccagt gggtgctcac 420 tgccgcccac tgcttcttcg tgacccgggagaaggtcctg gagggctgga aggtgtacgc 480 gggcaccagc aactgcacca gttgcctgaggcagctccat tgccgagatc atcatcaaca 540 gcaattacac cgatgaggag gacgactattgacatcgccc tcatgcggct gttccaagcc 600 cctgaacctg tccgtcacat ccaccctgcttgcctccccc atgcatggac agacctttag 660 cctcaatgag acctgttgga tcacaggctttggcaaagac agggagacag atgaaaagac 720 atcccccttc ctcgggaggt gcaggtcaatctcatcgact tccagaaatg caatgactaa 780 ctggtctatg acagtacctt acccaaggatgatgtgtgtg gggaacttcg tggg 834 29 621 DNA mouse 29 agatcatcat ctgccaggtcagcctccacg acatcctccc caacgagagt gtaccttgtt 60 agagcaacac cagtgggggctgtccccatc cgggcatctc ctgccaggtc agcaccagcc 120 accagggcca ccagggtagagcccaggtct cagtttcccc aagttctcct ggtcaggaga 180 cccagaggca gctgccactcatcgggtgtg tcatccttct catcagcctg gtgatctcgc 240 tcatccttct cttctacttctggagagtgc cacacaggga tcaagtacaa agagccactg 300 gagagttgcc ctatccacgcagttcgctgt gatggagtgg tggacttgca aaatgaagag 360 cgatgagctg ggctgtgtcaggttcgactg ggacaagtcc ctcctgaaag tctactctgg 420 gtcttctggc agagtggcttcctgtctgca gcagcagcgg aacgacactg actccaagag 480 gacctgccag caagctgggatttgacagcg cttaccgaac aactgaggta gcccacagag 540 acatcaccag cagcttctaactctcggaaa caaaacaaca tccaggaaag gctctacagg 600 tcgaatgtct tccggcggat g621 30 678 DNA mouse 30 tcagcctcca cgacatcctc cccaacgaga gtgtaccttgttagagcaac accagtgggg 60 gctgtcccca tccgggcatc tcctgccagg tcagcaccagccaccagggc caccagggag 120 agcccaggtc tcagtttccc caagttctcc tggcaggagacccagaggca gctgccactc 180 atcgggtgtg tcatccttct catcagcctg gtgatctcgctcatccttct cttctacttc 240 tggagaggcc acacagggat caagtacaaa gagccactggagagttgccc tatccacgca 300 gttcgctgtg atggagtggt ggactgcaaa atgaagagcgatgagctggg ctgtgtcagg 360 ttcgactggg acaagtccct cctgaaagtc tactctgggtcttctggcga gtggcttcct 420 gtctgcagca gcagctggaa cgacactgac tccaagaggacctgccagca gctgggattt 480 gacagcgctt accgaacaac tgaggtagcc cacaggaacatcaccagcag cttcttactc 540 tccgaataca acaccaccat ccaggaaagc ctctacaggtcgcaatgtcc ttccggcggt 600 atgtctccct ccagtgttcc cacgtggttt ggagctatgacgggcggacg aggaggggtc 660 gacctcgaag catgcctg 678 31 577 DNA mouse 31aagttttgat tacgcgcttt ctgcaattga tctcttgtta tttaaaccaa cggtttcagg 60tcaatctttg gagtatttgt agcttctaat ttttgaaatg actgaattaa gaatttggat 120gcttgctctt ttggttggtt tgcctaaaat ccagcccaca atccagtcgt ctcttgggag 180agggaggtgc cttgcaaact ttcatataac gaatgtgcct gaggctgctt aactctggac 240tagtctcaga tctcaaacct gcactacacg aggaggcata cttttgcttc atctggacat 300ttagaatact gtaaccttgc tgccgttctg ttagattgct aactacgtcc cccgtctcca 360atttggctct ccttaggcga taggatttgt cgtttttaac ggcaataaac ttgacaacac 420cagaatccaa gttttacttg aaaagctcgg cagaatacac agtggtgtga caaaaaacaa 480cagcaaaggg ttcctttgtg caatgacaaa cggtaaaaat gctgtaacgt tgaagaataa 540ctatttccac gcaagaacct cctgcttgac tgtgtat 577 32 688 DNA mouse 32ggtgatctcg ctccatccgt tctcttctac ttctggagag tgccacacac gggatcaagt 60acaacggagc cactggagag ttgccctatc cacgcagttc gctgtgatgg agtggtggac 120tgcaaaatga agcagcgata gagctgggct gtgtcaggtt cgactgggac aagtccctcc 180tgaaagtcta ctctgggtct tctggcgagt ggcttcctgt ctgcagcagc gagctggaac 240gacactgact ccaagaggac ctgccagcag ctgggattct gacagcgctt accgaacaac 300tgaggtagcc cactagagac tgtcaccagc agcttcttga ctctccgaat acgacaccac 360caatccagga aagcctctac aggtcgcaat atccttcccg gcggtaatgg tctcccatcc 420agtgttccca ctgtggtttg agagcctatg accgggcgga tcgtgggagg cggctctgaa 480cctcggagag caagtgcgcc ctggctaagt tagcctgcac ttcggcaact acccacattc 540tgtggcggca cacttcatcg atagcccagt gtgttctcca ccggttgcca ccgttttttg 600tgaccccgca acaacctctt aacaagtgac aacacctttt tccaccacaa atgtcccacg 660acccacaagt ccttctcccc aactcttg 688 33 614 DNA mouse 33 ccagatcatcatcaacggca actacacaga tgaacaggat gactatgaca ttgccctcat 60 caggctgtccaagcccctga ccctgtcagc tcacatccac cctgcctgcc tcccgatgca 120 cggtcagaccttcggcctca atgagacctg tggatcacgg gcttggcaaa accaaggaga 180 cagatgagaagacatctccc ttcctccgag aggttcaggt caacctcatt gacttcaaga 240 agtgcaatgactacttggtc tatgacagct accttacccc aaggatgatg tgtgccgggg 300 atctacgaggagggagggac tcctgccagg gagacagtgg aggacctctc gtctgtgagc 360 agaacaatcgctggtacctg gcaggtgtca ccagctgggg cacaggctgt ggccagaaaa 420 acaagcctggtgtgtacacc aaagtgacag aagtacttcc ctggatttac agaaagatgg 480 agagtgaggtacgattccgg aaatcttaac catgtcctcc tcacgtagct gactgctatg 540 aagatcctgggcacagggat ggggccattt gcagccatct ggtacagtgg acaacaagca 600 cctttggttctccc 614 34 751 DNA Homo sapiens 34 aagcctggag gactcttccc ctcagagaccatggagaggg acagccacgg gaatgcatct 60 ccagcaagaa caccttcaga ctggagcatctccagcccag gcatctccag ctgggacacc 120 tccaggccgg gcatctccag cccaggcatcactttccagg tcctcatcct ggcaggtcat 180 catccgccag gtcagcctcg gtgacaacctccccaaccag agtgtacctt gttagagcaa 240 caccagtggg ggctgtaccc atccgatcatctcctgccag gtcagcacca gcaaccaggg 300 ccacagtgga gagcccaggt acgagcctgaccaagttcaa ctgagcaggg agggccagaa 360 gcagctaccg actcatcgga gtgcagtgctcactcctcat tgccctggat ggtttacgct 420 catcatcctc ttccagttct ggcagggcacacagggatca aggtcacaag gagcaagatg 480 tgtgagagct tgtcccaaag cacgcctgttcgcttgtgca cggggtgtat gggacttcca 540 aagactgaag aggtgacaga cgctgtgctagcgtgaggta ttgactggga ccaacgtctc 600 tgctttaaaa tcttactctg ggtccctccaatcagtggga tcccatctgt agcagcacct 660 gggaattgac tctactacag agaagactgccagcgagtgg gatcaaagag gtccccggga 720 cacgaggtgg ccacaggatt ggcaaagatt a751 35 1230 DNA Homo sapiens misc_feature (1)...(1230) n = A,T,C or G 35atgacccagc tgtctgcttc tttttctcta gtccagttct ggcagnncca cacagnnatc 60aggtacaagg agcagaggga gagctgtccc aagcacgctg ttcgctgtga cggggtggtg 120gactgcaagc tgaagagtga cgagctgggc tgcgtgaggt ttgactggga caagtctctg 180cttaaaatct actctgggtc ctcccatcag tggcttccca tctgtagcag caactggaat 240gactcctact cagagaagac ctgccagcag ctgggtttcg agagtgctca ccggacaacc 300gaggttgccc acagggattt tgccaacagc ttctcaatct tgagatacaa ctccaccatc 360caggaaagcc tccacaggtc tgaatgccct tcccagcggt atatctctct ccagtgttcc 420cactgcggac tgagggccat gaccgggcgg atcgtgggag gggcgctggc ctcggatagc 480aagtggcctt ggcaagtgag tctgcacttc ggcaccaccc acatctgtgg aggcacgctc 540attgacgccc agtgggtgct cactgccgcc cactgcttct tcgtgacccg ggagaaggtc 600ctggagggct ggaaggtgta cgcgggcacc agcaacctgc accagttgcc tgaggcagcc 660tccattgccg agatcatcat caacagcaat tacaccgatg aggaggacga ctatgacatc 720gccctcatgc ggctgtccaa gcccctgacc ctgtccggtg agggaatctg cactccccgc 780tctcctgccc cccagcccca gcaccctctg cagccctcgc acttgtcagc atctgtcaac 840tcatatccgg gccccaaagc ttctgcagac aagacatccc ccttcctccg ggaggtgcag 900gtcaatctca tcgacttcaa gaaatgcaat gactacttgg tctatgacag ttaccttacc 960ccaaggatga tgtgtgctgg ggaccttcgt gggggcagag actcctgcca gggagacagc 1020ggggggcctc ttgtctgtga gcagaacaac cgctggtacc tggcaggtgt caccagctgg 1080ggcacaggct gtggccagag aaacaaacct ggtgtgtaca ccaaagtgac agaagttctt 1140ccctggattt acagcaagat ggaggcgagg tgcgattcag aaaatcctaa ccagctggcc 1200tgctgctctg cacagcaccg gctgctgtga 1230 36 24 DNA Artificial SequenceRandom oligonucleotide 36 tcaactgact agatgtacat ggac 24

1-30. (canceled)
 31. A method of screening for agents that can regulatean activity of a human transmembrane serine protease, comprising thesteps of: contacting a test compound with a polypeptide comprising anamino acid sequence selected from the group consisting of: (a) the aminoacid sequence shown in SEQ ID NO:12, (b) the amino acid sequence encodedby a cDNA insert contained within plasmid pCRII-TMSP3 (ATCC AccessionNo. PTA-3433), and (c) biologically active variants thereof; anddetecting binding of the test compound to the polypeptide, wherein atest compound that binds to the polypeptide is identified as a potentialagent for regulating the activity of the human transmembrane serineprotease.
 32. The method of claim 31 wherein the step of contacting isin a cell.
 33. The method of claim 32 wherein the cell is in vitro. 34.The method of claim 32 wherein the cell is in vivo.
 35. The method ofclaim 31 wherein the step of contacting is in a cell-free system. 36.The method of claim 31 wherein the polypeptide comprises a detectablelabel.
 37. The method of claim 31 wherein the test compound comprises adetectable label.
 38. The method of claim 31 wherein the polypeptide isbound to a solid support.
 39. The method of claim 31 wherein the testcompound is bound to a solid support. 40-73. (canceled)